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How the oxygen tolerance of a [NiFe]-hydrogenase depends on quaternary structure

‘Oxygen-tolerant’ [NiFe]-hydrogenases can catalyze H2 oxidation under aerobic conditions, avoiding oxygenation and destruction of the active site. In one mechanism accounting for this special property, membrane-bound [NiFe]-hydrogenases accommodate a pool of electrons that allows an O2 molecule attacking the active site to be converted rapidly to harmless water. An important advantage may stem from having a dimeric or higher-order quaternary structure in which the electron-transfer relay chain of one partner is electronically coupled to that in the other. Hydrogenase-1 from E. coli has a dimeric structure in which the distal [4Fe-4S] clusters in each monomer are located approximately 12 Å apart, a distance conducive to fast electron tunneling. Such an arrangement can ensure that electrons from H2 oxidation released at the active site of one partner are immediately transferred to its counterpart when an O2 molecule attacks. This paper addresses the role of long-range, inter-domain electron transfer in the mechanism of O2-tolerance by comparing the properties of monomeric and dimeric forms of Hydrogenase-1. The results reveal a further interesting advantage that quaternary structure affords to proteins.

Introduction
Hydrogenases, microbial metalloenzymes that produce or oxidize H 2 , are important for understanding the energy balance and metabolism of microoganisms, in addition to being inspirational models for future hydrogen catalysts [1,2]. The active sites of the two main classes, known as  Figure 1 provides structural information that will be relevant for this paper. So-called 'standard' [NiFe]-hydrogenases consist of a large (α) subunit containing the [NiFe]active site and a small (β) subunit containing a relay of FeS clusters to transfer electrons. These air-sensitive enzymes have been extensively investigated by crystallography and spectrosocopic techniques with significant input from molecular biology strategies [2]. Attached to an electrode, hydrogenases are extremely efficient electrocatalysts, and protein film electrochemistry (PFE), in which catalytic rate is measured directly as current, has made important contributions to our understanding of their functional properties [3][4][5].
A special sub-category of [NiFe]-hydrogenases, known as O 2 -tolerant hydrogenases, have the special property of displaying sustained activity in the presence of O 2 [6][7][8][9][10]. These hydrogenases operate because they destroy O 2 by rapidly converting it to water, thus avoiding reactive oxygen species that would otherwise oxygenate the active site and render it inactive for long periods-a state/states known as 'Unready' or 'Ni-A' [10][11][12]. Under H 2 , membrane-bound O 2 -tolerant hydrogenases react with O 2 to form a Ni(III)-OH complex, known as Ni-B ('Ready'), that is rapidly re-activated by reduction to an active Ni(II) species with release of the OH ligand. An efficient electron supply thus serves two roles-(a) securing complete reduction of O 2 , by-passing reactive intermediates, and (b) ensuring rapid re-activation of Ni-B (regarded as a resting state). The structures of 'standard' (strongly O 2 -inhibited) [NiFe] hydrogenases, such as D. fructosovorans hydrogenase (Fig. 1a) [1,13], have been known since 1995, but only recently have the structures of O 2 -tolerant hydrogenases of the subgroup of membrane-bound respiratory [NiFe]-hydrogenases (MBH) isolated from Ralstonia eutropha H16, Hydrogenovibrio marinus, Escherichia coli and Salmonella enterica, been established [14][15][16][17]. These new structures have not only revealed a novel [4Fe-3S] cluster, able to transfer two electrons sequentially [9,[15][16][17][18], but they also show interesting quaternary organization that may also be important in conferring O 2 -tolerance, as discussed below. The MBHs not only contain additional membrane-domain subunits that dissociate upon isolation with detergents, but also exist as oligomers of the minimal αβ 'heterodimer'.
The crystal structure of Hydrogenase-1 (Hyd-1) from E. coli shows it to be a (αβ) 2 dimer, in contrast to the (αβ) monomer of standard hydrogenases (Fig. 1a, b) [16]. The  . This structure (PDB: 4GD3) of the P242C variant with a P-to-C exchange at the medial FeS cluster also shows a cytochrome subunit that is absent from other crystal structures. c The redox centers in Hyd-1: [NiFe] active site and iron sulfur clusters labeled with edge-to-edge electron transfer distances. The position of the b-type heme in the membrane anchor is also shown (from the structure of P242C) structure supports earlier determination of the molecular mass of Hyd-1 as 200 ± 20 kDa, [19]. It was also known that in vivo, Hyd-1 associates with membrane-intrinsic subunits that contain cytochrome b [19,20]. Although the first crystal structure obtained for Hyd-1 did not include its cognate cytochrome b subunit, a subsequent structure obtained for P242C variant (depicted in Fig. 1b) included the membrane-intrinsic subunits in which one cytochrome b remained attached [21]. In the isolation of solubilized Hyd-1, the cytochrome b subunits are normally lost during the homogenization stage.
The distance between the two distal clusters in each half of Hyd-1 is only 12.2 Å. It has been proposed that an intersite distance below 14 Å is generally short enough to allow electron tunneling to occur at a sufficient rate that catalysis is not limited [22]. Comparing the distances between the other iron-sulfur clusters in Hyd-1 (ca. 12.2 vs. 7.7 Å, 10.6 and 11.1 Å Fig. 1c) therefore suggests that electron exchange between the two distal clusters and thus between the two (αβ) halves should be feasible. Shomura and coworkers, having solved the similarly heterotetrameric structure of the membrane-bound hydrogenase (MBH) from H. marinus, ruled out the possibility that dimerization might be an artifact of crystallization, on the basis that the contact between heteromers adds up to 11 % of the total surface area of the protein and the subunits have precisely matching orientations-the latter observation pointing towards purposeful joint alignment on the cell membrane [15].
In an unusual experiment that shed further light on the importance of long-range intermolecular/interfacial electron transfer in O 2 tolerance, Wait and co-workers measured the power characteristics of a membrane-less fuel cell with two carbon electrodes, one modified with Hyd-1 (the H 2 -oxidizing anode) and the other with bilirubin oxidase (the O 2 -reducing cathode) [23]. Such an investigation is an electrochemical experiment without a source of potential control or external electrons. The O 2 -tolerant hydrogenase allows the fuel cell to operate with a non-explosive H 2 -air mixture; however, use of a weak H 2 mixture (3 %) resulted in loss of power, apparently irreversibly, when the load resistance was set sufficiently small to collapse the voltage. The 'short circuit' caused the oxidizing power due to O 2 reacting at the cathode to be transmitted directly to the anode: as a result, and without enough H 2 to act as counterbalance, Hyd-1 was converted rapidly to Ni-B. Power could not be restored by increasing the resistance, but momentarily connecting a second anode with active Hyd-1 resulted in immediate recovery. This effect was likened to jump-starting a car that has a flat battery, with active hydrogenases on the repairing electrode providing electrons to reactivate the hydrogenases on the fuel cell anode that have been completely converted to the Ni-B state. Volbeda and co-workers suggested that the proximity of the distal clusters in the two halves of Hyd-1 might enable a similar 'jump-start' reactivation of Ni-B to operate internally in the (αβ) 2 dimer [21].
Oligomer formation can be predicted using PISA (proteins, interfaces, structures and assemblies) software which calculates energies and entropies of dissociation into monomers [24]. Table 1 shows an assessment of current [NiFe] hydrogenase structures, evaluated with Pymol and PISA. All the O 2 -tolerant hydrogenases are membrane-bound in vivo and all but the R. eutropha MBH show dimers of heterodimers (αβ) 2 in the crystal structure, although it was reported that R. eutropha MBH may form a trimeric (αβγ) 3 supercomplex including cytochrome b, based on a static light scattering analysis [25]. For O 2 -tolerant hydrogenases the free energy of dissociation (ΔG diss ) within the (αβ) heterodimer is twice as large as the corresponding interheterodimer (αβ) values (60 vs. 30 kcal/mol), and higher than for standard hydrogenases. The hydrogenase from A. vinosum is the only standard hydrogenase found to crystallize as a dimer, albeit with only half the calculated ΔG diss . The distance between the two distal FeS clusters, averaging around 12.5 Å in the O 2 -tolerant enzymes, is higher in the A. vinosum hydrogenase, at 14 Å.
An interesting observation was reported recently, in which the full (αβγ) 3 complex of R. eutropha MBH was studied by PFE, utilizing a tethered lipid bilayer to immobilize the enzyme on a gold electrode [26]. Unlike the normal soluble form that has been extensively studied by PFE at a graphite electrode, the (αβγ) 3 complex did not inactivate anaerobically when poised at a high potential, and re-activation after exposure to O 2 occurred spontaneously even at high potentials when O 2 was removed.
The question is therefore raised-what role could extended inter-heterodimer electron transfer play in protecting a hydrogenase against O 2 ? Such a protection role would represent a further example of the importance of quaternary structure in biology [27]. We have now investigated the significance of the (αβ) 2 structure of solubilized Hyd-1 in relation to hydrogenase O 2 -tolerance. This required a systematic step-by-step strategy-establishing how to obtain the monomeric (αβ) form and separate it from the (αβ) 2 dimer, investigating the stability and catalytic properties of the (αβ) form, comparing the O 2 -tolerances of (αβ) and (αβ) 2 , and finally devising a model that accounts for the differences.

Materials and methods
The general procedures for obtaining purified Hyd-1 from E. coli cells were based on those previously established [6]. Samples were stored in liquid N 2 as required. Size exclusion chromatography (SEC) columns were calibrated with the Sigma Aldrich MWGF1000 kit of protein standards. Since the elution volume corresponding to a certain molecular mass depended significantly on the detergent used, and varied with each repacking, the column was recalibrated each time for each detergent. Alcohol dehydrogenase (ADH) protein standard (150 kDa) was used between sample runs to help evaluate sample peak positions and confirm column integrity.
Protein samples for native electrophoresis were prepared by addition of sample buffer (Invitrogen, final concentration 50 mM BisTris, 6 N HCl, 50 mM NaCl, 10 % w/v glycerol, 0.001 % Ponceau S, pH 7.2). NativeMarkTM Unstained Protein Standard (Invitrogen) was used as a reference. The Anode Buffer consisted simply of Running Buffer (50 mM BisTris, 50 mM Tricine, pH 6.8) while the Cathode Buffer included 20× Cathode Additive (0.4 % Coomassie r G-250). The gel (pre-cast 4-16 % BisTris, Invitrogen) was run at 150 V for ca. 130 min, using the XCell SureLockTM electrophoresis system (Life Technologies). The gel was developed as follows: incubation in ca. 100 mL fixing solution (40 % methanol, 10 % acetic acid) for approx. 60 s in the microwave at ca. 700 W, followed by 15-20 min at room temperature on a gel shaker. Subsequently the gel was transferred to destaining solution (8 % acetic acid) and incubated, first in the microwave for 60 s at 700 W and then at room temperature on the shaker until satisfactory.
Protein samples for denaturing electrophoresis were prepared by addition of 4× LDS sample buffer (Invitrogen) (total volume 10 μL) and heating for 10 min at 70 °C. PageRuler ® pre-stained protein ladder (Thermo Scientific) was used as a reference standard. The samples were loaded onto a NuPAGEr 4-12 % Bis-Tris pre-cast gel (Invitrogen) and run at 200 V for ca. 50 min in MOPS running buffer (50 mM MOPS, 50 mM Tris base, 1 mM EDTA, 0.1 % SDS, pH 7.7) using the XCell SureLockTM electrophoresis system (Life Technologies). Gels were developed by incubation in Coomassie staining solution (0.25 % Coomassie brilliant blue, 50 % ethanol, 10 % glacial acetic acid, deionized water) for ca. 10 min at a temperature between 50 and 60 °C followed by subsequent destaining until satisfactory (20 % ethanol, 10 % glacial acetic acid, deionised water).
Procedures for isotope ratio mass spectrometry and hydrogen peroxide assays were carried out as described recently [11]. Protein film electrochemistry methods, including voltammetry and chronoamperometry were based on experiments described in the recent paper by Evans et al. [7]. All reagents used to prepare samples for solution assays or protein film electrochemistry were of analytical grade and high-purity water (Milli-Q, Millipore 18 MΩ cm) was used throughout. All gases were supplied by BOC.  [24], the oligomeric assembly of large subunit α and small subunit β is presented along with PISA estimates of the free energy of assembly dissociation (ΔG diss ) in kcal/ mol of large and small subunit within one heteromer (α:β) and between two heteromers

Results
Separation of oligomeric states of Hyd-1 Figure 2a shows a Hyd-1 oligomer separation carried out in buffer containing 0.02 % Triton X-100. Soluble aggregate is found near the void (around 8-9.5 mL). A large, well-defined peak at 12 mL and a much smaller peak at 13.5 mL, corresponding to molecular masses of approximately 220 and 110 kDa, respectively, are assigned to dimer and monomer forms of Hyd-1. Significant absorbance at 420 nm due to iron-sulfur clusters and heme is found for both aggregate and the dimer peaks. The inset (Fig. 2a) shows an SEC analysis of the dimer fraction after 5 days storage at 4 °C. The fact that most protein elutes at the same volume (12 mL) suggests that the dimeric form is the stable state. However, the reproducibility of the separation experiment with Triton X-100 (major panel Fig. 2a) was poor, especially with regard to the monomer, the low yield of which is clear from analysis of the isolation products by Native gel electrophoresis, as shown in Fig. 3a. The elution profile depended further on how isolated Hyd-1 is stored. Replacing Triton X-100 with digitonin, a detergent used to purify R. eutropha membrane-bound hydrogenase (MBH) in a fragile multimeric complex with its native cytochrome b 562 , led to more reliable isolation of Hyd-1 monomer [25]. The best results were obtained by pre-incubating freshly purified samples in buffer containing ca. 1 % (wt/vol) digitonin then using just 0.007 % digitonin in the running buffer to minimize detergent precipitation. The molecular masses of the protein peaks identified as 'Dimer' and 'Monomer', calculated relative to a full calibration, are 210 kDa and 104 kDa, respectively. Particularly notable is the presence of two distinct A 420 peaks pairing with the A 280 dimer and monomer peaks.
To evaluate the subunit composition of the aggregate and putative dimer and monomer peaks isolated from Triton X-100 and digitonin SEC experiments (Fig. 2b/c), denaturing electrophoresis was carried out (Fig. 3b). All samples show characteristic large (64.6 kDa) and small (36.8 kDa) subunit bands just below the 70 and 40 kDa marker bands, demonstrating that the monomer and dimer fractions (at approximately 110 and 220 kDa total mass) are structurally intact and consist of at least one and two assemblies, respectively, of a large and small subunit each. Some lanes are slightly overloaded (widening of the large subunit band) which is somewhat unavoidable in the search for fainter bands. While the overloading prevents a truly reliable quantitative comparison of band density, the Triton X-100 aggregate appears to have a particularly low proportion of small subunit. Hyd-1 size exclusion chromatography with Triton X-100 and digitonin: all samples shown were first purified by Ni-NTA affinity chromatography. The absorbance, at 420 and/or 280 nm, is shown across the elution volume. a Separation of Hyd-1 with Triton X-100, exchanged from Ni-NTA purification buffer (20 mM Tris, 350 mM NaCl, 0.02 % Triton; see "Materials and methods") into running buffer prior to the experiment. The peak at 12 mL was isolated and stored at 4 °C for 5 days before being subjected to chromatography again, as shown in the inset. b Separation of defrosted Hyd-1 sam-ples from the liquid nitrogen dewar and a −80 °C freezer, with Triton X-100. The samples were thawed immediately before applying to the column. c Freshly isolated Hyd-1 was incubated for 2 h with ca. 1 % digitonin before chromatographic separation in running buffer containing 0.007 % digitonin. The arrow and asterisk mark the peak position of 150 kDa alcohol dehydrogenase (ADH) protein standard. Other conditions: flow rate 0.15 mL/min, superdex 200 HR column (10/30), pH 7.0, 50 mM sodium phosphate, 0.10 M NaCl, 4 °C. The column was repacked between experiments a, b and c

Reactivation and O 2 reduction in solution
Hydrogen oxidation by hydrogenases in solution is easily measured by monitoring the increase in absorbance at 604 nm (A 604 ) due to enzymatic reduction of benzyl viologen in the presence of H 2 [6]. When using enzyme samples that have previously been activated under H 2 then exposed to air to cause inactivation, a lag phase in which there is no change in A 604 is commonly observed at the beginning of the assay, when all the viologen is in the oxidized state. The lag phase arises because enzyme reactivation depends on the supply of electrons (transferred to viologen) originating from H 2 oxidation by those sites that are active: there are no electrons available at the start of the experiment, and few active enzyme molecules to generate them.
The lag phases of dimer and monomer samples were compared. Initial hydrogen oxidation assays indicated that dimer samples were more than twice as active as monomer, based on protein concentrations determined by Bradford assay. However, the Bradford assay is very susceptible to, and easily biased by, the presence of detergents, that are essential for sample preparation, storage and use. Reagent binding might also be significantly affected by the level of protein aggregation. Determination of protein concentration by UV-vis spectroscopy (A 280 ) yielded turnover rates that were closer in value. To compare different measurements in the light of this uncertainty, the amount of enzyme used in the eventual solution-based reactivation assays was adjusted to yield samples of equal final activity and not apparent protein concentration. Figure 4a shows a typical result of the lag phase experiment, in which previously activated dimer and monomer samples were exposed to air for several hours before the assay. Immediately after injection into H 2 -saturated benzyl viologen buffer, the absorbance at 604 nm was recorded over time. The eventual rate of viologen reduction in the samples was adjusted to be as similar as possible; in this case approx. 5.9 μM s −1 for the dimer and 6.7 μM s −1 for the monomer solution. The lag phases differ greatly between monomer and dimer. Figure 4b compares the lag phases observed in three sets of experiments, carried out using enzyme from three separate preparations. Although both overall activity and length of lag phase vary between different Hyd-1 preparations, the difference between dimer and monomer for each individual preparation was clear and consistent: the lag phase of the monomer is always slightly more than twice as long as that of the dimer.
Oxygen-18 reduction experiments for dimer and monomer samples were carried out using isotope ratio mass spectrometry to detect the amount of H 2 18 O formed. The methodology was as described recently [11]. Due to the limited amount of monomer available, the following protocol was used. First, the concentration of dimer samples was determined by Bradford assay, then the absorbance at 280 nm (A 280 ) was recorded by UV-vis spectroscopy to obtain a molar absorption coefficient from which the concentration of monomer samples could also be determined. The concentration of enzyme is expressed with reference to the functional monomer containing one active site and having a mass of 101 kDa. Figure 5 shows the results obtained from eighteen data points in Ref. [11] where an H 2

18
O formation rate of 0.65 s −1 was obtained; this compares with rates obtained (three datum points) from samples incubated for 4, 10 and 14 h for dimer (0.78 s −1 ) and monomer (0.67 s −1 ). Long incubation times were needed to accumulate enough products for mass spectrometry measurements, due to Hyd-1 monomer concentrations being as low as 0.06 μM.

Protein film electrochemistry of dimer and monomer
To evaluate any obvious electrochemical differences between dimer and monomer forms of Hyd-1, cyclic voltammetry was performed on enzyme films grown using the two different fractions on a pyrolytic graphite edge (PGE) electrode using published procedures [3]. Figure 6a shows cyclic voltammetry experiments carried out with monomer and dimer forms. The potential was scanned at 1 mV s −1 and the PGE electrode was rotated at 3000 rpm, conditions found to give steady-state voltammetry free of time dependence or mass-transport limitations. The line shows the average current for forward and reverse scans. The dimer yielded significantly higher catalytic current than the monomer (an approximate fourfold difference being typical) irrespective of whether the film was obtained through application of more or equally concentrated dimer solution relative to the monomer. Only in experiments with very dilute monomer samples (a symptom of very low monomer yields in early isolation experiments) were larger differences in total current observed. The plot in Fig. 6a initially suggests that the monomer has a more positive onset potential (the potential at which the catalytic current begins to rise) so a normalization procedure was devised to allow a better comparison of the two results. Figure 6b compares the first derivative (slope) of the monomer and dimer voltammograms, which locates a characteristic potential at which the rate increases most steeply. The potentials of maximum rate of current change are very similar for monomer and dimer, at approximately −203  O per μM Hyd-1) compared to blank controls is given for `as isolated' Hyd-1 and resolved dimer and monomer forms. The error bars show the standard deviation across 18, 3 and 3 measurements, respectively. The concentration of H 2 18 O was measured by isotope ratio mass spectrometry as described previously [11]. The experiments were conducted under an atmosphere of 90 % H 2 and 10 % 18 O 2 at pH 7.0 and 20 °C and −212 mV vs. SHE, respectively. By normalizing the voltammograms to the currents at these potentials, Fig. 6c was obtained: importantly, H 2 oxidation by both oligomeric forms of Hyd-1 shows a nearly identical response at the low-potential edge where both show the same onset potential of approx. −335 mV vs. SHE at the conditions used. With the H 2 /H + equilibrium potential E eq at −391 mV vs. SHE, the onset overpotential requirement is approximately 56 mV for both oligomeric forms.
At higher potential however, the two voltammograms differ significantly. Three factors principally affect the catalytic current of Hyd-1 at high potentials: (1) At sufficiently high currents, the current could be limited by H 2 mass transport; this factor was eliminated by the use of 1 bar H 2 and a rotation rate of 3000 rpm (after establishing that a higher rotation rate did not result in an increase in current).
(2) Dispersion of interfacial electron-transfer rates, due to the various orientations that can be adopted by enzyme molecules on an electrode surface, gives rise to a residual current instead of the flat plateau that is normally expected once electron transfer is no longer limiting [28]. (3) Slow oxidative conversion of the enzyme into the inactive Ni-B state. A procedure was devised to assess the potential at which Ni-B is stable. The Hyd-1 film on the electrode was subjected to a high-potential poise (+0.42 V vs SHE) for sufficient time to convert much of the sample to Ni-B, then the potential was scanned in the negative direction at a very low scan rate, e.g. 0.1 mV s −1 . In such an experiment, shown in Fig. 6d, the data were normalized in the same way as those in panel C, and the two traces overlay Fig. 6 Protein film voltammetry of dimer and monomer forms of Hyd-1: a averaged forward and backward scans (scan rate 1 mV s −1 ) of dimer and monomer films on a rotating PGE electrode. b First derivative of the averaged cyclic voltammograms from panel a. c Voltammograms normalized with respect to the current at the potential of the maximum derivative. d Linear sweep scans of dimer and monomer films, slowly swept from +0.42 to −0.44 V vs. SHE at 0.1 mV s −1 , to monitor reactivation of the anaerobically inactivated oxidized state (Ni-B). As in panel c, the voltammograms are normalized to the current at the potential of maximum derivative. Common conditions: 100 % H 2 , pH 6.5, electrode rotation rate 3000 rpm very well at low potential. Although the two voltammograms differ at high potential because the fraction of monomeric enzyme that has inactivated is smaller (the extent of anaerobic, electrochemical inactivation may depend on the proficiency of electronic coupling between enzyme and electrode) the re-activation potential referred to as E switch (an empirical reference point for the re-activation process) is altered very little.
The hallmark of oxygen tolerance is sustained activity in the presence of O 2 . Mass spectrometry results have shown that Hyd-1 achieves this through rapid reduction of O 2 to water, which results only in Ni-B [11]. Chronoamperometric experiments on Hyd-1, in which the H 2 oxidation current at a given potential was monitored during each addition and subsequent removal of O 2 from the gas supply, showed that for each O 2 concentration, the current settled at a new stable level, equivalent to O 2 behaving as a reversible inhibitor [7]. A simple term for the fractional (steady-state) activity f was introduced, f being the steady-state H 2 oxidation current for a given O 2 level relative to that observed in the absence of O 2 . It was proposed that f depends on the rates of inactivation r I and reactivation r A to/from the Ni-B state according to Eq. 1: Chronoamperometry experiments were carried out to determine how the oligomeric state of Hyd-1 affects the steady-state activity observed in the presence of different amounts of O 2 . The experiment was conducted at 10 mV vs. SHE, a potential sufficiently high to have a good H 2 oxidation current but, equally, negative enough to avoid anaerobic Ni-B formation. In Fig. 7a, the H 2 oxidation rate under 100 % H 2 was allowed to stabilize for 400 s,

Discussion
Use of freshly isolated Hyd-1 and digitonin proved not only to allow the clearest, most reliable separation of distinct dimer and monomer fractions, but also produced the least wastage in the form of aggregate. Although initially stable and soluble, aggregate fractions were observed to give substantial precipitate over time. Obtaining sufficient quantities of monomer was the chief challenge for a rigorous characterization of the monomer fraction, since only the trailing side of each monomer peak could be used to avoid isolating a heterogeneous sample. Applying very concentrated samples to a gel filtration column, in an effort to reduce the number of purifications needed to accumulate enough enzymes to study, tended to be futile, since loading concentrated samples lowers the peak separation. The (αβ:αβ) dissociation free energy of 31.8 kcal/mol (Table 1) for Hyd-1 is likely an overestimate since the underlying calculation does not account for the presence of detergent. From gel electrophoresis, both dimer and monomer as well as larger aggregate fractions consist of both small (36.8 kDa) and large (64.6 kDa) subunits. The ca. 210 and 104 kDa oligomers are thus confidently assigned as dimer (αβ) 2 and monomer (αβ).
The onset overpotential of approximately +56 mV (pH 6.5, 100 % H 2 and 30 °C) and the current response to increasing potential around the onset potential are similar for both monomer and dimer. Onset overpotential requirements for `as-isolated' Hyd-1 were previously reported as approximately +50 mV under 10 % H 2 (pH 6.0, 30 °C) by Lukey et al. [6], or in a more detailed study by Murphy and co-workers as +54 mV and +82 mV at pH 6.0 and pH 7.0, respectively (100 % H 2 , 37 °C) [4]. The values determined here for the pure oligomeric fractions agree well with these earlier measurements, noting that the onset potential increases with decreasing pH [4].
Differences in the voltammetry traces recorded for dimer and monomer are most prominent at high potential. The more pronounced residual current (slope) for monomer may reflect the ability of the (newly) solvent-exposed area in the monomer (i.e. the interface area in the dimer) to offer a greater range of interactions and orientations with the electrode surface. Importantly, the potential E switch , a measure of the stability of Ni-B, is unchanged, and in conjunction with the similar onset overpotential, supports the idea that the Hyd-1 monomer is fully functional.
A particularly striking difference between monomer and dimer forms of Hyd-1 was displayed in the non-electrochemical, solution assays of H 2 oxidation, where it was established that the initial lag phase for the monomer is more than twice as long as for the dimer (Fig. 4b). Questions arising are: (1) does the dimeric organization help in ensuring exclusive formation of rapidly reactivated Ni-B, and does the monomer therefore produce less-easily reactivated Unready states (Ni-A) ?. We noted earlier that Hyd-1 activity in solution is maintained at a constant level in 10 % O 2 , an observation that argues against Ni-A formation [11]. (2) Does the dimeric organization affect the rates of reactivation of inactive states? (3) Can a result similar to the difference in lag phase be reproduced in a more controlled environment, where uncertainty over enzyme concentration is not important? The chronoamperometry experiments, in which Hyd-1 attains a steady state with simultaneous substrate (H 2 ) and competing substrate/ inhibitor (O 2 ) turnover, were helpful in answering these questions.
The facts that both dimer and monomer attain a steadystate H 2 oxidation activity in the presence of O 2 (Fig. 7) and recover activity fully when O 2 is removed show that the ability to exclusively form Ni-B (and no Ni-A) and thus reduce O 2 completely to water are unaffected by the oligomeric state of the enzyme. Formation of even a small fraction of Ni-A would lead to a persistent and largely irreversible decrease in catalytic current. Even under 10 % O 2 the almost zero activity of the monomer recovers virtually completely when the O 2 is removed from the gas stream. The main difference in all cases is that the fractional activity is significantly diminished for the monomer compared to the dimer. Further analysis of these steady-state levels holds the key to developing a plausible mechanistic explanation of the effects of dimerization.
Using the steady-state description introduced above, rates are substituted by rate constants: and assuming r I = k I [O 2 ] at the low [O 2 ] values used (Evans et al. measured the initial rate of O 2 reaction with active Hyd-1 and found it to increase linearly with O 2 concentration [7]), f at any given potential is given by: Figure 8a shows a fit to Eq. 2 for the fraction of active dimer at each O 2 concentration. The fit used a ratio C of apparent reactivation to inactivation rate constant of C D /μM = k A /k I = 40 (at +10 mV). For the monomer, no fit could be obtained with any combination of rate constants; the best approximation was C M /μM = k A /k I = 15 with k A being 38 % that of the dimer.
Although the individual rate constants cannot be obtained directly from these experiments, previous experiments with Hyd-1 provide a guideline. Wulff [7]. From an Arrhenius plot, the rate constant for inactivation at 25 °C is estimated as k I = 0.0028 (μM O 2 ) −1 s −1 . With C D /μM = k A /k I = 40 (see above) the rate constant for reactivation is deduced to be k A = 0.112 s −1 for the presented fit to the dimer data. An interesting point, however, is that k A is strongly potential dependent (see below). Since the reactivation rate is very fast at low potentials, it is not directly measurable under experimental conditions chosen to allow significant H 2 oxidation activity at high O 2 concentrations. This problem was addressed previously by Evans et al. [7] by extrapolation from data obtained at both higher potential and lower temperature to the desired conditions, with the help of electrochemical activation and Arrhenius plots. Using the same process, rate constants of k A = 0.136 s −1 and k A = 0.186 s −1 were calculated for −10 mV vs. SHE and 25 °C from two separate data sets in the paper by Evans et al.; the discrepancy between these two values illustrates the uncertainty and deviations introduced by double extrapolation. These calculations showed that the fit to the dimer data is achieved The blue text represents enzyme that is active in H 2 oxidation. Note that formation of E from B may also require the presence of H 2 [29] with rate constants that agree reasonably well with predicted values.
The simple model shown in Scheme 1 assumes that O 2 attacks the active enzyme (E) when the active site is in the highest active oxidation state and all the FeS clusters are reduced. In the Ni-B state (B) that is rapidly formed, three electrons have been transferred from the FeS relay leaving one site (the distal cluster) still reduced. State B can be reactivated rapidly, starting with reduction of the active site Ni back to Ni 2+ by intramolecular electron transfer, but the fact that k A is a potential-dependent rate constant suggests that we should regard the FeS clusters of the relay collectively as acting like a low-level tunneling barrier-a 'wire' along which electron transfer is accelerated by a potential difference. The enzyme now re-enters the catalytic H 2 cycle through binding of H 2 and subsequent turnover [29]. Binding of H 2 to the active site in the Ni 2+ state to re-establish E is assumed to occur on a much faster time scale than the relatively slow reaction with O 2 , and is thus considered to be effectively instantaneous. After an indefinite number of H 2 turnovers, another O 2 may attack, and active enzyme E may enter another cycle of the slower O 2 catalysis.
The simple model for the reaction of Hyd-1 with O 2 was now reconsidered to see if any improvement could be made. An extension of the model needed to account for the observation that the active monomer fraction decreases more strongly than the active dimer fraction when the O 2 concentration is increased; hence two further mechanistic features were introduced. First, it had been noted previously that a relatively small but significant superoxide/peroxide producing side reaction is observed for Hyd-1 in the presence of O 2 [11]. Second, we considered the likelihood that the two partners in a dimer might be able to share electrons via the distal clusters. These considerations yielded the extended reaction scheme included in Scheme 1.
The first stage ocurs as before: O 2 attacks an active enzyme molecule E to give Ni-B (B in Scheme 1) in a fourelectron reaction that produces two molecules of water. If we assume that all the FeS clusters are reduced before O 2 attacks, two electrons are delivered from the proximal FeS cluster P, one stems from the medial cluster M and the final electron results from oxidation of the active site Ni, such that the distal cluster D can remain reduced. Protons are omitted from this scheme which also aids simplicity. Under steady-state conditions B is best described as (Ni 3+ -P OO M O D R ). Formation of a Michaelis complex E:O 2 in the initial reaction of active enzyme with O 2 was also considered but discarded, as a fit to the data simply required a very large K m value to impose the same linearity as in the simpler model.
The extended model includes the possibility that inactive enzyme B may react in an alternative manner and become more oxidized, exhausting the FeS relay system of electrons. The electron residing in the FeS relay system that is normally available to transfer to the active site could transfer instead to another O 2 molecule. Such a site is likely to be the distal cluster that lies closest to the protein surface and able to undergo an outer-sphere reduction of O 2 to superoxide O 2 − , resulting in a fully oxidized enzyme O that requires external electrons for re-activation. The source of this electron depends on the particular experiment (electrochemical vs solution), whether monomer or dimer is present, and on the rate constant k red for electron transfer to the distal cluster, specifically, either between subunits (k DD ) or interfacial (k ED ). It is very likely that the electron must tunnel over a longer distance from the electrode to the distal cluster than between the distal clusters, so that k DD ≫ k ED . Thus, even on an electrode the dimer should have an advantage in O 2 tolerance, since at any point in time it is likely to have at least one reduced distal cluster. Superoxide formation at the distal cluster could result in damage, although in the electrochemical experiment, O 2 − would be removed rapidly by electrode rotation.
Another possible pathway for reactivation is the very slow reaction (k a ) of fully oxidized enzyme O with H 2 to activate the enzyme, a process that accounts for the lag period in solution assays when no external electron donor is present. There is one precedent for this possibility, in which slow, direct reaction of Ready (Ni-B) with H 2 is reported [30]. In the electrochemical experiment, when compared to reduction of the distal cluster by long-range electron transfers, k a should be largely irrelevant for the dimer; hence, the overall rate of (dimer) reactivation v A from B to E can be simplified by analogy to a reversible inhibition (Eq. 3) depending on [O 2 ] with an apparent inhibition constant K C proportional to k red /k ox : Incorporation of v A in place of k A in Eq. 2 yields the fraction of active enzyme for the new extended model: Incorporating this extension into the model improves the fit to the data at high O 2 concentration even for the dimer (see Fig. 8b). The large value for K C used for the dimer (K C = 1000) implies that distal cluster reduction via the distal-distal pathway is very fast compared to k ox , so that the oxidizing side reaction has a small effect which only becomes discernible at high O 2 concentrations (where coinciding oxidation of both distal clusters in a dimer also becomes more probable). In the monomer, oxidation of the (4) f = Active enzyme Total enzyme = . distal cluster in state B leaves only the slower k ED contribution to k red and a much lower value for the constant K C is expected. The slowest pathway for reactivation k a might also play a small role under these circumstances. In agreement with these considerations, a good fit to the monomer data is obtained using the new extended model with K C = 20 as shown in Fig. 8b. Two further points are worth noting. The potentialdependence of the observed k A values [7] is also consistent with the effect of electrode potential on distal cluster oxidation state and reverse supply of electrons k ED for reactivation, as discussed above. The isotope ratio mass spectrometry experiment also supports the idea that the differences in O 2 tolerance between dimer and monomer do not originate from separate pathways for primary O 2 attack (k I ).
The excellent fit of the new extended model to the data for both dimer and monomer (Fig. 8b) is a strong argument for its validity. On a statistical basis, attacks by O 2 molecules during normal H 2 turnover are likely only to affect one half of the enzyme at a given instant unless the O 2 concentration is very high. Rapid sharing of electrons between distal clusters for delivery to the active site, as described by the extended model, also explains why the lag phase in solution assays is much longer for monomer. According to Scheme 1, the lag phase is due to the very slow direct reaction with H 2 (k a ). For the dimer, only half of all active sites need to be reactivated via this slow pathway, explaining the observed ca. two fold difference in lag phase between dimer and monomer (Fig. 4). Once enzymes have become activated, electrons are available through the build up of reduced viologen and the process accelerates.
In conclusion, we have demonstrated that it is possible to separate distinct oligomeric states of E. coli Hyd-1. Specifically, an (αβ) 2 dimer of heterodimers and an (αβ) monomer of heterodimers were isolated. The dimer is very stable, easy to isolate and is the favored species at increasing detergent concentrations where larger aggregate fractions are broken down. Experiments carried out to investigate the functional advantages that a dimer structure confers led to increased O 2 tolerance as being the most significant property. The mechanism is complicated but we have proposed that O 2 tolerance depends in some way on the ability to transfer electrons between the distal clusters in each (αβ) monomer half. Just as the explanation for the fuel cell experiment lay in an analogy with jump-starting a car with a flat battery, the normal function of Hyd-1, which is to catalyze H 2 oxidation in the face of regular attacks by O 2 , depends upon the constant presence of a partner to provide a rescue electron when needed. Electron transfer between each half of the dimer is more rapid than interfacial electron transfer at modest electrochemical driving force. In conclusion, teamwork pays off even in biological electron transfer!


In-situ spectroscopic studies of viologen based electrochromic device

Fabrication and operation of simple solid state electrochromic devices using ethyl viologen diperchlorate in a polymer matrix is presented here. In-situ Raman and transmission/absorption studies have been done to establish the origin of bias induced color change, between a transparent and navy blue color, in the electrochromic device. The origin of bias induced color change has been attributed to the bias induced redox switching between its viologen dication and free redicle forms. Fundamental reason behind colour changes of viologen molecule has been established. In-situ UV-Vis spectra reveals that the navy blue color of the device under biased condition is not due to increase in the transparency corresponding to blue wavelength but due to suppression of the transparency corresponding to the complementary colors. Absorption modulation has been reported from the device with good ON/OFF contrast of the device.

Introduction
An electrochromic device, as name suggests, is a device which changes color as a result of an electrical bias. This phenomenon of bias induced color change, called electrochromism, was first observed in the nineteenth century [1]. Electrochromism has been studied in recent times because of many applications in science and technology. [2][3][4][5] Detailed reviews are available in the literature about electrochromic effects from different chemicals and materials. [2,6] Electrochromic effects are observed as a result of redox processes either in a solid-state device or in an electrochemical cell. Origin of this property lies in the fact that many materials show multiple redox states with different optical properties (e.g. absorption spectra). Different electrochromic materials are used as the active material, which could be in the form of metal oxide like tungsten oxide [7] or a cationic molecule. [8][9][10] One particularly intriguing class of materials in this context is bipyridinium species, which are formed upon N,N-diquaternization of 4,4'-bipyridine, also known as viologens. An example of such a violgen molecule is shown in scheme-1. Viologen is an organic compound for potential use in flexible electrochromic devices due to its activity in reversible redox reaction and excelent electron accepting nature. In other words, Viologens [9], [11][12][13][14] are reducible materials used in solid-state electrochromic devices, which may undergo one-or two-electron reductions. [9], [11][12][13][14] Ease of processing and use have stimulated intensive research into the electrochromic properties of viologens. The current research was undertaken to better understand viologen electrochromism and to simplify the device design for realization of a solid-state electrochromic device.

Scheme 1: Ethyl Viologen molecule
Controlling the often complex stoichiometry of metal oxides is a complication when fabricating electrochromic devices based on these materials [15,16]. In addition, many metal oxides lack sufficient transparency for use in a wide range of applications. In contrast, viologen based materials are solution processable and can yield flexible electrochromic devices. In order to achieve an electrochromic response in the solid state, an ionically conducting matrix is required as well as a redox counter-reaction.
Very recently, lithium perchlorate in polyethylene oxide (PEO) has been used successfully as a solid electrolyte for organic electronics in general and thin film transistors in particular [10], [17][18][19][20][21]. Ethyl viologen in PEO matrix can be a good choice for making viologen based eletrochromic devices, at least for testing purposes. Ethyl viologen diperchlorate (EV (ClO 4 ) 2 ) can be a good candidate because it contains EV 2+ which is a good electron acceptor and also contains perchlorate ion which can act as electrolyte. Its solution in an appropriate solvent is transparent and does not absorb visible light. After accepting an electron it gets reduced and form radical cation EV + . In the reduced state it shows absorption peak at 396 and 606nm which indicate blue color of the device [22]. The advantage with viologen is that it shows reversible switching between the two oxidation states mentioned above. In other words, EV + can go back to EV 2+ by oxidation, which shows no absorption in the visible region which is transparent state of the device. Details of redox reactions taking place during oxidation and reduction of EV have been provided in SI separately. A solid state electrochromic device can be made using viologens by appropriately controlling the redox process using an electric bias. Many of the viologen applications are largely based on its ability to change in the electron absorption spectrum by reversible oxidationreduction reaction. This feature of viologen can be tuned by altering the substituents on the nitrogen and used it for applications spanning from photochemistry and solar energy conversion, molecular wire and switches. Recently viologen has been used as single molecular transistor Viologen is used as chemical quencher for improvement of quantum yield of nanoemitter [23]. Viologens are frequently used as electrochemical label, photochemical probe or in studies on the electron-transport processes or oxidative damage in DNA [24].
In the present work a simple electrochromic device has been fabricated in two geometries to study the fundamental reason of colour change. A clear understanding may enable one to get other colors than blue. In depth studies have been done by observing bias induced changes in EV species using spectroscopic techniques in-situ. The in-situ experiments helps in understanding the underlying mechanism for bias induced color change which is required for making a versatile electrochromic device. Nondestructive spectroscopic techniques like Raman and UV-Vis may prove to be handy in analyzing the mechanism for the same. The two geometries used to understand the bias induced color change enables us to carry out in-situ measurements for an in-depth analysis. In the simplest geometry, the EV (in an appropriate matrix) has been sandwiched between two Indium Tin Oxide (ITO) electrodes on quartz substrate to get the device in cross-bar geometry (CBG). Similar device was fabricated by drop casting EV in between two gold electrodes separated by few  Cross-bar geometry (CBG) device has been fabricated by placing a film of EV+PEO between two trans- parent conducting electrodes of ITO deposited on quartz substrate using magnetron sputtering deposition [25]. The EV+PEO film was obtained on an ITO coated substrate using spin coating. After spin coating, second ITO electrode was laminated face to face on the spin coated substrate. The extreme part of both the electrodes was painted by silver glue for making connection with external power supply. UV-Vis spectroscopy was performed using Cary 60 UV-Vis spectrophotometer of Agilent whereas Raman spectroscopy was done using LABRAM HR spectrometer using a 633 nm excitation source. Figure 1 is the combined pictorial representation of internal and external mechanism of CBG device. Fig. 1(a) shows cross sectional schematic view of CBG device with its biasing arrangement. Fig.1(b) is corresponding actual photographs of CBG device on various voltages. Fig 1(c) corresponds to proposed model which shows molecular dynamics and redox reaction within the device as a result of applied bias. Fig.1(a) shows that when external potential is 0 V, device is in its OFF state which corresponds to the transparent state and thus 'IITI'

Results and Discussion
can be seen through the device clearly in Fig. 1(b). The transparency of the complete device reduces and becomes opaque after the application of -3.5 V bias as shown in Fig.1(b). Color change in viologen is a well reported phenomenon [26] and is often assigned to the different absorption/transmission properties exhibited by viologen species in its different oxidation states. Typical optical properties of chemically reduced viologen show that blue color originates due to the presence of EV + species which can be obtained by chemically reducing EV 2+ (present in ethyl viologen diperchlorate) which is transparent. Alternatively, similar redox When potential is applied, electrons are supplied by the power supply and infusion of electron take place into the compound which may be reducing the viologen molecules near the -ve electrode to get the opaque (ON) state when most of the viologen molecule gets reduced. EV 2+ and ClO 4− species (in PEO matrix) are randomly distributed in between the two ITO electrodes in the absence of bias (V=0). When bias is applied (V = -1V) electrons supplied by the ve electrode is accepted by EV 2+ to get reduced to EV + to initiate the color change. As higher voltages are applied more reduction take place and large number of EV + gets generated which results in opaque device. In addition, the perchlorate and EV + ions move towards positive and negative electrodes respectively. As a result, complete opaque nature of device is obtained with arrangement of ions in the device as shown in the case of V = -3.5V bias case in Fig. 1(c).
Alternatively, it may also be possible that bias induced color change is associated with the (electric) field induced structural change [27] of the molecule which may lead to band gap variation and hence change in optical properties [28]. To discount this effect control experiment was done by sandwiching viologen between two quartz substrates without the ITO electrode. Schematic for this experiment has been shown in Fig S2 in the SI. The present biasing condition does not provide electrons to the molecule (for possible reduction) but do provide field for any field-related structural change. No color change was observed upto a bias voltage of 50V which suggests that the observed color switching is not related to field induced structural change and more likely reason being the transmission change due to redox process.
The blue color visible as a result of bias in Fig. 1 has only qualitative meaning and needs more scientific approach to identify the presence of different wavelength components in the transmitted light when the device is seen in a visible light. Perceived color by human eye must be cross checked with scientific results (with optical tools) before making any conclusions. To understand the color changing phenomena in CBG device the best suitable technique is Ultra-violet visible (UV-Vis) spectroscopy. From UV-Vis spectra one can understand the actual wavelength of light which is more affected by the reduction of viologen molecule. Insitu UV-Vis spectroscopy has been performed to investigate optical property of the device which are clearly visible from naked eyes. Figure 2 represent transmission spectra with respect to change in bias voltage using UV-Vis spectroscopy. Transmittance (%) of the film depends upon the band-gap of the material so the band-gap modulation is carried out by the application of voltage During in-situ measurement, bias was increased gradually upto -3.5V(in steps of 0.5 V) and transmittance of the device is measured in the range between 300 to 800 nm. The transmission spectrum under zero bias condition has been recorded as reference data, to see any variation in it after bias application. Under no bias condition, only ITO and EV 2+ (in PEO matrix) species are present and a peak appears around 560 nm. The peak around 560 nm gets suppressed as the biasing potential is increased and finally it changed into a valley shaped curve for a voltage of -3.5 V. It can be noticed from Fig.2 that there is not much change in %T at the wavelengths of blue (425 nm) and red (725 nm) colours as compared to the wavelengths in between these two. The device exhibited a minimum transmittance in the visible region at around 590 (orange-yellow) nm with a transmittance of 22 % at -3.5V. As a result, the counter colour of orange-yellow, i.e, blue, is observable to the visible eyes. This also means that blue color of the device in ON state is not due to the enhancement of blue wavelength but due to suppression of other colors in the visible range.
It is clearly observed from the transmittance spectrum corresponding to -3.5V that transmission is suppressed in the range of 500 to 700 nm. Also, from the color wheel we can say the counter colour of most suppressed colour in transmission spectra will be the colour of the film [29]. The simple analysis of UV-Vis spectra allows one to understand the origin of the color appearance of the electrochromic device. This also may enable one to understand that % T corresponding to a given wavelength may be tuned by appropriately enhancing/suppressing the %T spectra at other wavelengths. Relative %T corresponding to different wave-  Figure 3 shows the CIE color calculator chart with the photograph of our actual devices with different applied bias. The CIE color calculator has been used here to calculate CIE coordinates corresponding to the device appearance color for a given applied bias. Initially at zero bias (OFF) device is transparent hence it allow the white light to pass through it [31]. As the applied bias voltage increase, only blue color spectrum pass through it as compared to green and red. According to colour appearance, related point is observe on different coordinate of the chart [32] as shown in Fig. 3. Finally it gets saturated at dark blue colour and their corresponding coordinate on CIE chart are (0.16, 0.12). Hence except blue color, device absorbs all colors in its ON state. The CIE chart is very useful as it says that if somehow the material, to be used in device, is tuned in such a way that the color corresponding to coordinates (0.5,0.5) is allowed to transmit from the device a red color appearance will be observed. If one identifies the species responsible for appearance of one particular color corresponding to a given coordinate, the same can be used to make a devise giving that particular color. Before implementing this idea, it is very important to understand the species responsible for a particular color's appearance. This investigation can be done using Raman scattering experiments. Figure 4 shows Raman spectra of the device in its OFF and ON states. Raman spectrum of the device in its OFF state is similar to the EV(ClO 4 ) 2 +PEO mixture in powder form. This means that the device contains the EV 2+ form of viologen in the OFF state. Raman spectrum of the device in the ON state is different from its OFF state with some new peaks appearing and some disappearing. This certainly means that the species present in the ON and OFF states, giving rise to the Raman peaks, are different. It is also likely that the peaks appearing in the ON state are obtained as a result of reduction of the species present in the OFF state. To confirm this Raman spectra in Fig. 4 have been compared with the Raman spectra from EV 2+ and EV + as obtained by chemically reducing the EV(ClO 4 ) 2 using NaBH 4 which is shown in Fig.  S3 in the SI. Comparison of Fig. 4 and Fig. S3 shows that the OFF device contains EV 2+ whereas the ON device contains EV + in majority which means that EV 2+ reduces to EV + as a result of applied bias more likely at the ve electrode. This cannot be confirmed in the CBG because a spatial Raman spectroscopy is not possible.
To confirm this, in-situ Raman spectroscopy of the device in OFG has been carried out. Device in OFG was prepared by drop casting the active material (EV+PEO) on a pre-fabricated electrode system where two gold electrodes are a few microns apart. A drop of prepared sample in the gap completes electrical circuit and device will start working like electrochemical cell. Top view of the finished device is shown in Fig. 5(a) without and with bias applied. The polarity scheme of the power supply to give bias is shown in Fig. 5(b), which shows the cross-sectional schematic of the device. A color change on electrode E1 as a result of ve bias on this electrode can be seen which is similar to the bias induced colour change in Fig. 2 . This can be appreciated by comparing point X and Y in Fig. 5(a). Raman spectra has been recorded from points X,Y and Z (Fig. 5(a) and on E 1 electrode changed which attributes reduction of EV 2+ molecule.
The corresponding Raman scattering studies have been done on E 1 and E 2 . Alphabets X, Y, Z on the microscopic images indicate the positions from where the Raman signals were collected. Raman spectrum in figure 5(c) X was recorded from the E 1 terminal of of an unbiased device, which shows major peak at 932 cm 2+ , the peaks corresponding to EV 2+ . Raman spectra 5(c)Y and 5(c)Z have been recorded under bias of -3.5V on E 1 and E 2 respectively. Some additional peaks appear on the E 1 electrode in biased condition, spectrum 5(c)Y shows two prominent peaks corresponding to reduced viologen which are at 1028 and 1528 cm −1 . With biasing potential, peak intensity is increases as the applied potential increases which shows the formation of more free radical cations (EV + )There is no change in the spectrum recorded on E 2 where +ve terminal of the power supply is connected.
For the comparative study of various peaks positions, Raman spectra of neutral and chemically reduced form of EV(ClO 4 ) 2 has been carried out to verify spectra obtain from electrode E 1 and E 2 under different bias conditions (Fig. 5(c)). To identify and verify [33] the changes in EV 2+ , EV + and EV o concentration Raman peaks of chemically reduced state of EV were studied. The solid EV(ClO 4 ) 2 powder was reduced using an aqueous solution of sodium borohydride (NaBH 4 ). Raman spectrum from this reduced state of EV(ClO 4 ) 2 is shown in Fig S3 in SI and it was observed that the EV powder turns dark-bluish immediately after mixing with NaBH 4 due to reduction of EV. This change of colour is due to variation in absorption spectrum. The same solution turns back into transparent due to oxidation of EV + to EV 2+ as higher oxidation state is the more stable state in the ambient conditions. Raman spectrum from chemically reduced form of EV(ClO 4 ) 2 shows peaks at 1028 and 1528 cm −1 which are signatures of EV + species.(These peaks are marked by stars in figure S4). It is now safe to conclude that the blue color observed in Fig. 2 as a result of applied bias is due to the EV + species being generated at the ve terminal as a result of reduction of EV 2+ . By combining the data in Figs 2, 5 & 3S, one can see that the Raman spectrum from the dark region on the device (E 1 electrode in Fig. 5(a)) shows the presence of EV + species whereas the virgin device shows the signature of stable EV 2+ . Therefore, the Raman spectra permit the conclusion that the mechanism of bias induced color change in the electrochromic device is due to formation of free radical cation from viologen dication.
Above discussion confirms the origin of color change in the electrochromic device. It will be interesting to see how fast and how many times the electrochromic device be turned ON and OFF. Figure 6 shows ON and OFF cycles in terms of normalized absorbance corresponding to 580 nm which has been studies within a specific time period [34]. Initially at 580 nm, on zero bias absorbance is in its lowest value, Al which reaches the highest value Ah by applying potential (-3.5V) making it opaque for this wavelength. Figure 6 shows that device can be turned ON and OFF five times in ∼100 seconds. It is worth mentioning that it takes more time in returning to OFF state after removal of the applied bias because the returning to OFF state is natural and is not under any external perturbation. A 50 % transparency in the second cycle onwards has been considered as OFF state whereas ON state corresponds to 100 % transparency. Additionally, contrast between the ON and OFF states can be calculated by defining colour contrast ratio (CCR) using the following equation [35][36][37].
Here T i and T f are the initial and final values of transmittance for the device at wavelengths 580 nm.
The values of T i and T f are 80% and 12% giving a 68% variation in transmittance and CCR of nearly 85%. Figure 6 also shows that it takes approximately 1.5 seconds to switch between the ON and OFF states of the device. An electrochromic device with 85% CCR is a very good device with an ability to switch in 1.5 seconds which can be improved if an external method is used for turning OFF the device of course by modifying the device composition and geometry appropriately.

Conclusions
In-situ spectroscopic studies of viologen based electrochromic devices reveal that bias induced color change appears due to bias induced change in oxidation state of viologen. The blue color of the device as a result of negative bias is obtained due to presence of viologen frees redical (EV + ). Raman spectra from the devices under this bias condition confirms the presence of viologen free redical in both the geometries of device. Raman signature corresponding to the free redical is seen on the electrode connected to the negative polarity of the power supply (used to bias the device) whereas EV 2+ (viologen dication) is present on the other electrode which was connected to the positive polarity. Species on the positive terminal of the device is same as the species present in the whole device in its virgin state. Since the cross bar geometry device is symmetric, it shows color switching between transparent to navy blue aster application of bias irrespective of the polarity. In-situ UV-Vis spectra reveals that the navy blue color of the device under biased condition is not due to increase in the transparency corresponding to blue wavelength but due to suppression of the transparency corresponding to the complementary colors. A 85 % contrast and 68 % variation in color has been observed in the device


extfieldsofstudy: Chemistry Medicine

text:
Difluoromethanesulfonyl hypervalent iodonium ylides for electrophilic difluoromethylthiolation reactions under copper catalysis

Difluoromethanesulfonyl hypervalent iodonium ylides 2 were developed as electrophilic difluoromethylthiolation reagents for a wide range of nucleophiles. Enamines, indoles, β-keto esters, silyl enol ethers and pyrroles were effectively reacted with 2 affording desired difluoromethylthio (SCF2H)-substituted compounds in good to high yields under copper catalysis. The reaction of allyl alcohols with 2 under the same conditions provided difluoromethylsulfinyl (S(O)CF2H) products in good yields. The difluoromethylthiolation of enamines is particularly effective with wide generality, thus the enamine method was nicely extended to the synthesis of a series of difluoromethythiolated cyclic and acyclic β-keto esters, 1,3-diketones, pyrazole and pyrimidine derivatives by a consecutive, two-step one-pot reaction using 2.

General Information
All reactions were performed in oven-dried glassware under a positive pressure of nitrogen. Solvents were transferred via syringe and were introduced into the reaction vessels through a rubber septum. All solvents were purified by standard method. All of the reactions were monitored by thin-layer chromatography (TLC) carried out on 0.25 mm Merck silica gel (60-F254). The TLC plates were visualized with UV light or KMnO 4 in water/heat. All of the reaction products were purified by Column chromatography. Column chromatography was carried out on a column packed with silica gel 60N spherical neutral size 63-210 mm. The 19 F NMR (282 MHz) and 1 H NMR (300 MHz) in CDCl 3 , (CD 3 ) 2 CO or (CD 3 ) 2 SO were recorded on a Varian Mercury 300. 13 C NMR (125 MHz) spectra for solution in CDCl 3 , CD 3 CN or (CD 3 ) 2 SO were recorded on a BRUKER Advance 500. Chemical shifts () are expressed in ppm downfield from internal TMS (= 0.00) or C 6 F 6 [= -162.2 (CDCl 3 ) or -163.5 ((CD 3 ) 2 CO)] as an internal standard. Coupling constants (J) are in Hertz (Hz). The following abbreviations were used to explain the multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad. Mass spectra were recorded on a SHIMADZU GCMS-QP5050A (EI-MS) and SHIMAZU LCMS-2020 (ESI-MS and APCI-MS). Infrared spectra were recorded on JASCO FT/IR-200 or a JASCO FT/IR-4100 spectrometer.

Preparation of Reagent 2a-d and Starting Materials
Synthesis of Reagent 2a-d Scheme S1. Synthesis of reagent 2a-d

Synthesis of reagent 2a:
A mixture of 2-bromo-4'-nitoroacetophenone (2.00 g, 8.2 mmol) and sodium difluoromethanesulfinate (1.25 g, 9.1 mmol) in DMAc (70 mL) was stirred at 50 ºC for 20 h. Then water (50 mL) was added to the mixture at room temperature. The resulting mixture was extracted with Et 2 O and organic layer was washed with water 2 times, brine for once then dried over magnesium sulfate. The solvent was removed by rotary evaporation to give a crude product then the crude product was purified by frash column chromatography on silica gel (eluent: ethyl acetate/hexane =1/1, Rf = 0.5 -0.6). A yellow solid (1-(4-

Experimental Details (A) General procedure for difluoromethylthiolation of enamines 3, indoles 5 and pyrroles 7.
To a mixture of enamine 3, indole 5 or pyrrole 7 (0.2 mmol) and Cu(I)Br (0.04 mmol) in 1,4-dioxane (2.5 mL), reagent 2a (0.4 mmol) or 2d (0.4 mmol) was added at room temperature under N 2 . The resulting mixture was stirred at room temperature for 5 h and then filtered through a short plug of celite. The filtrate was concentrated under reduced pressure and purified through flash column chromatography on silica gel (ethyl acetate/hexane) to afforded the target product.

(B) General procedure for difluoromethylthiolation of -keto esters 9
To a mixture of -keto ester 9 (0.2 mmol) Cu(I)Br (0.04 mmol) and K 2 CO 3 (0.08 mmol) in 1,4-dioxane (2.5 mL), reagent 2d (0.4 mmol) was added at room temperature under N 2 . The resulting mixture was stirred at room temperature for 24 h and then filtered through a short plug of celite. The filtrate was concentrated under reduced pressure and purified through flash column chromatography on silica gel (ethyl acetate/hexane) to afforded the target product.
(C) General procedure for reaction of ally alcohols 11 with reagent 2.
To a mixture of ally alcohol (0.20 mmol) and CuF 2 (0.04 mmol) in DMAc (2.5 mL), reagent 2a (0.4 mmol) was added at room temperature under N 2 . After stirring at room temperature for 24 h, water (2.0 mL) was added to the mixture. The resulting mixture was extracted with ethyl acetate, the organic phase was washed with water 2 times and saturated sodium bicarbonate solution over 5 times to remove the starting material and brine once then dried over magnesium sulfate. The solvent was removed by rotary evaporation and purified through flash column chromatography on silica gel (ethyl acetate/hexane) to afforded the target product. After stirring at room temperature for 5 h, 1N-HCl aq (1.0 mL) was added to the reaction mixture. The resulting mixture was continuously stirred for 12 h (for 24 h when 3q or 3r was a starting material), then extracted with ethyl acetate, the organic phase was washed with water 2 times and brine once then dried over magnesium sulfate. The solvent was removed by rotary evaporation and purified through flash column chromatography on silica gel (ethyl acetate/hexane) to afforded the target product. To a mixture of -keto esters (0.2 mmol), CuF 2 (0.04 mmol) and Potassium carbonate (0.04 mmol) in DMAc (2.5 mL), reagent 2d (0.4 mmol) was added at room temperature under N 2 . After stirring at room temperature for 18 h or 16 h. The resulting mixture was then extracted with ethyl acetate, the organic phase was washed with water and brine once then dried over magnesium sulfate. The solvent was removed by rotary evaporation and purified through flash column chromatography on silica gel (DCM/hexane) to afforded the target product.
(F) General procedure for reaction of silyl enol ether 9d with reagent 2.
To a mixture of silyl enol ether (0.20 mmol) and CuBr (0.04 mmol) in 1,4-dioxane (2.5 mL), reagent 2d (0.4 mmol) was added at room temperature under N 2 . After stirring at room temperature for 18 h, 1 N HClaq (1.0 mL) was added to the mixture and stirring at room temperature for 30 min. The resulting mixture was extracted with ethyl acetate, the organic phase was washed with water 2 times and brine once then dried over magnesium sulfate. The solvent was removed by rotary evaporation and purified through flash column chromatography on silica gel (DCM/hexane) to afforded the target product. Reagent 2a-d (0.1 mmol, respectively) was put into a glass tube at room temperature in air atmosphere. After 1 h, 5 h, 10 h and 24 h respectively, CDCl 3 and PhF (0.2 mmol: an internal standard) were added to the resulting reagent. The residual rate of reagent was immediately analyzed by 19 F NMR spectroscopy.

Scheme S2. Reaction of 3m with Reagent 2a under Different conditions
(1): To a mixture of enamine 3m (0.1 mmol) and Cu(I)Br (2.9 mg, 0.02 mmol) in 1,4-dioxane (1.3 mL), reagent 2a (174.5 mg, 0.2 mmol) was added at room temperature under N 2 . The resulting mixture was stirred at room temperature for 5 h and then filtered through a short plug of celite. The filtrate was concentrated under reduced pressure then analyzed by 19 F NMR spectroscopy with PhF (18.4 L, 0.2 mmol) as an internal standard.
(2): The mixture of Cu(I)Br (2.9 mg, 0.02 mmol), 2a (174.5 mg, 0.2 mmol) and 1,4-dioxane (1.3 mL) was stirred at room temperature for 20 min under N 2 . 3m was added to the resulting mixture and then stirred for 5 h. The reaction mixture was filtered through a short plug of celite. The filtrate was concentrated under reduced pressure then analyzed by 19 F NMR spectroscopy with PhF (18.4 L, 0.2 mmol) as an internal standard.

Scheme S3. GCMS Analysis of Difluoromethylthiolation of 3m with 2d
To a mixture of enamine 3m (18.9 mg, 0.1 mmol) and Cu(I)Br (2.8 mg, 0.02 mmol) in 1,4-dioxane (1.3 mL), reagent 2d (104.7 mg, 0.2 mmol) was added at room temperature under N 2 . The resulting mixture was stirred at room temperature for 5 h and then filtered through a short plug of celite. The filtrate 0.1 mL was added into CH 2 Cl 2 1.5 mL then the resulting mixture 5 L was analyzed by GCMS.
GCMS analytical data of the reaction mixture of 3m, CuBr and reagent 2d. All peak data (Peak 1-3)   atmosphere. b 19 F NMR yields with PhF as an internal standard.

4-(Difluoromethyl)thio-3-phenyl-1H-pyrazole (13)
To a mixture of enamine 3z (35.2 mg, 0.2 mmol, 1.0 equiv) and Cu(I)Br (5.7 mg, 0.04 mmol, 20 mol%) in 1,4-dioxane (2.5 ml), reagent 2a (174.5 mg, 0.4 mmol, 2.0 equiv) was added at room temperature. After stirring at room temperature for 5 h, hydrazine monohydrate (48.6 L, 1.0 mmol, 5.0 equiv) was added to the reaction mixture. The resulting mixture was continuously stirred at 90 °C for 5 h, then 2 mL of water was added and extracted with ethyl acetate, the organic phase was washed with water 2 times and brine once then dried over magnesium sulfate. The solvent was removed by rotary evaporation and purified through flash column chromatography on silica gel


Topical nano-delivery of 5-fluorouracil: Preparation and characterization of water-in-oil nanoemulsion

Purpose: To prepare and characterize a water-in-oil nanoemulsion of 5-fluorouracil (5FU) for enhanced skin penetration. Methods: Nanoemulsions of 5FU were prepared using Capyrol (propylene glycol monocaprylate). Transcutol (highly purified diethylene glycol monoethyl ether) and polyethylene glycol (PEG) 400 as oil, surfactant and co-surfactant, respectively. The optimized formulations were subjected to heating cooling cycling, centrifugation and freeze - thaw cycling to assess their stability. Particle size distribution and zeta potential of the nanoemulsions were evaluated. Furthermore, in vitro and in vivo skin permeation studies were carried out on the formulations in a rat model. Skin irritation studies were also performed on rats to assess the irritation potential of the formulations. The 1 % w/v of Carbopol 934 gel loaded with 1 % 5FU was used as control (FU gel). Results: The results showed that the mean droplet size of the nanoemulsions was ~100 nm with a zeta potential of ± 15. Significant increase in permeability was also observed for the nanoemulsion formulations compared with control. The steady-state flux (Jss), enhancement ratio and permeability coefficient (Kp) for optimized nanoemulsion formulation were significantly (p < 0.05) higher than those of the conventional gel (control). Both in vitro and in vivo skin retention results indicate higher drug release from the nanoemulsion (292.45 µg/cm 2 ) than for control (121.42 µg/cm 2 ). Mean irritation index for the nanoemulsion was significantly lower than for control. Conclusion: The results suggest that a water-in-oil nanoemulsion is safe and can potentially be used to promote skin penetration of 5FU following topical application on the skin for the treatment of some skin diseases.

INTRODUCTION
is an antineoplastic agent used for the treatment of skin diseases such as psoriasis, cutaneous premalignant, malignant lesions and basal-cell carcinoma [1]. However, in each instances less than optimal delivery of 5-FU using conventional preparations has necessitated the use of more invasive measures to obtain therapeutic outcomes. The ideal drug delivery for most skin diseases is for the drug to be able to penetrate deeper skin layers. As such the drug delivery strategy should be made to localize high drug concentrations inside the epidermis and dermis layers for effective therapy [2]. There has been an increased interest during recent years in the use of topical vehicle systems using permeation enhancers [3], but use of these chemical enhancers may be harmful, especially in chronic application. Therefore, topical vehicle systems could be made safer without chemical enhancers to facilitate drug permeation through the skin. One of the most promising formulation for enhancement of skin permeation of drugs is nanoemulsion [4].
Nanoemulsions are thermodynamically stable transparent (translucent) dispersions of oil and water stabilized by an interfacial film made by surfactant and co-surfactant molecules having a droplet size of less than 100 nm. The antiproliferative 5-FU has been extensively used as a topical chemotherapy for most skin diseases like cutaneous premalignant, malignant lesions and skin cancer (Basal-cell carcinoma [5]. Therefore, an improved 5FU nanoemulsion formulation with a high degree of permeation could be useful in the treatment various skin diseases. The present study describes the potential of nanoemulsion systems for topical delivery of 5FU that achieve the goal of increased skin permeation without the use of additional permeation enhancers.

Solubility studies
The solubility of 5FU in various oils was determined by adding excess amount of the drug in 2 mL of selected oils, surfactants and cosurfactants in 5mL vials, which were mixed thoroughly using a vortex mixer [6]. The vials placed in water bath -shaker for 24 h and the temperature was kept at 25 °C. After that, the mixtures were spun using a centrifuge (Bench Top Refrigerated, Eppendorf) at 3000 rpm for 15 min. The supernatants were sampled and quantified using a HPLC method. A Shimadzu HPLC system (Shimadzu, Japan) with two LC-10AT VP pumps with UV detector set at 260 nm and a RP C-18 column (150 mm x 4.6 mm I.D.; particle size 5 was used to quantify the 5FU content in solution.

Construction of pseudoternary phase diagrams
The ternary the pseudo-ternary phase diagram has been constructed based solubility of drug in each solvent (oil, surfactant and co-surfactant) [7]. The surfactant and co-surfactant were mixed in different ratios (1:1, 2:1, and 3:1). Phase diagrams were constructed for each ratio of mixture of surfactant -co-surfactant (S mix ) and oils. For every phase diagram, the proportion of oil and S mix varies in different volume ratio from 0:10 to 10:0. The percentage of the aqueous phase was determined using an aqueous titration method [8]. Slow titration of distilled water was added (5 % addition at a time) into the S mix -oil mixture and the observation of the transition from clear to turbid point was recorded. A calculation was made to determine the percentage of water, oil and S mix present at the point of turbidity. With the obtained individual percentage, a pseudoternary phase diagram was constructed with the clear-solution region marked and defined as the best emulsification region.

Preparation of nanoemulsion
From each phase diagram constructed, different points were selected from nanoemulsion region for incorporation surfactant, co-surfactant and oil. 1 % (w/w) of FU was dissolved in aqueous phase (distilled water) of all selected nanoemulsion formulation. Then, a selected quantity of S mix (mixture of surfactant and co-surfactant) was added into the aqueous solution of the drug. The mixtures were stirred for 2 min and sonicated until the drug dissolved completely. The oil phase (Capyrol PGMC) was added slowly with continuous stirring. The resulting mixtures were then passed through a high pressure homogenizer (Avestin Emulsifex C3) for 5 cycles.

Thermodynamic stability testing
The formulations were subjected to heatingcooling, centrifugation and freeze-thaw stability testing programme, where the physical appearances of the formulations were observed at each testing period. In the heating-cooling programme, all six formulations were heated at 45 °C and then cooled at 4 °C, with a duration of 24 h at each temperature, for 2 cycles. Then, formulations which passed the heating-cooling cycles were subjected to centrifugation at 3500 rpm for 15 min. Finally, only formulations which passed the previous two steps were stored at alternating temperature of -21 and 25 °C, with a duration of 24 h at each temperature, for 2 cycles.

In vitro skin permeation studies
Skin of 8-9 weeks old Swiss albino mice weighing 25 to 30 g was mounted in a Franz diffusion cell. The skin was washed with normal saline then dried between two filter papers. The skin was then carefully checked through a magnifying glass to ensure that samples were free from any surface irregularities such as tiny holes or cervices in the portion. 2 cm 2 of the excised skin samples were clamped between the donor and receptor chamber of a Franz diffusion cell with the stratum corneum facing the donor chamber. Then, 2 ml of prepared nanoemulsion formulations were administered into the donor chamber. The receptor chamber was filled with 15 ml of phosphate buffer solution (PBS) pH 7.4. The receptor medium was maintained at 37 ± 0.5 o C and stirred at 500 rpm throughout the experiment. 0.5 ml of sample was collected from the receptor medium of each cell every hour for 8 hours and then immediately replaced by the same amount of preheated PBS. The collected samples were filtered through a 0.45 μm pore size cellulose membrane filter and analyzed using a HPLC method as described above [9]. All animal protocols were approved by the Animal Ethics Committee of International Medical University, Malaysia (approval ref. no IAEC/BP/2012/0087). The animal experiment was performed in accordance with Directive 2010/63/EU on the handling of animals used for scientific purposes [10].

Permeation data analysis
The cumulative amount of drug permeated through the skin (µg/cm 2 ) was plotted against time in hours for each formulation. The drug flux (permeation rate) at steady-state (J ss ) and permeability coefficient (KP) were calculated from the slope of the linear portion of the graph using Eq 1 [11].
where, J ss is the drug flux at steady state and C 0 is the initial drug concentration in the donor cell. Enhancement ratio (Er) was calculated by dividing the J ss of respective formulation with J ss of control formulation as in Eq 2. Er = J ss F/J ss C ……………………………. (2) where, J ss F is the J ss of formulation and J ss C is the J ss of control.
The significance of results was checked statistically at p < 0.05 applying a one-way ANOVA test. Post hoc multiple comparisons were carried out using the least square difference test [12].
In vivo skin deposition studies 8-9 weeks old male Swiss albino mice weighing 20 to 25 g were used for this study. The animals were divided into 5 groups, with each group consisting of 5 animals. Prepared nanoemulsion FU2, placebo formulation (control), 5 % SLS and plain carbopol gel containing 1 % 5FU (plain gel) were applied to the dorsal surface (3 cm 2 ) of each group of mice after removal of its hair by clipping. The mice were then put to death by cervical dislocation at the end of 1, 2, 4 and 8 hours after dorsal application and the administered skins were stripped and adhering fats and debris were removed carefully. The skin was thoroughly washed with methanol and then dried. The drug content in the dermis skin layer was determined as described in the in vitro skin deposition study and drug content was analyzed by the HPLC method.

Skin irritation studies
Wistar albino rats (16 weeks) weighing 200 -250 g were used in this part of the study [12]. The animals were kept under standard laboratory conditions, with standard temperature of 25 °C ± 1 °C and relative humidity of 60 %. They had free access to a standard laboratory diet with water ad libitum. Prior to the application of formulations onto the animals, the hairs on the dorsal surface of the rats were removed by clipping. Five rats were assigned to each treatment groups: Group 1 received placebo formulation (negative control), Group 2 received sodium lauryl sulphate (SLS) solution (5 % w/v, positive control), Group 3 received prepared FU2 nanoemulsion formulation and Group 4 received carbopol gel containing 1 % 5FU. 200 µl of these prepared samples were applied onto the dorsal surface of the rats daily for six days using the reported method [13].
The rats were observed and erythema scores ranging from 0 to 4 were allocated depending upon the degree of erythema as follows, 0: No erythema 1: Slight erythema (barely perceptible light pink). 2: Moderate erythema (dark pink). 3: Moderate to severe erythema (light red). 4: Severe erythema (extreme redness).

Statistical analysis
Data are presented as mean ± standard (SD). The difference in results between batches containing various portion of excipients was statistically analyzed using Student's t-test with the aid of SPSS 16.0 statistical software. Differences were considered statistically significant at p < 0.05.

Solubility
In order to select the suitable solvents for the preparation of nanoemulsions, the solubility of 5FU various oil, co-surfactants and surfactants were determined and the results are shown in Figure1. The drugs that 5-FU has shown good solubility in are Trancutol HP, Capyrol PGMC and PEG 400 with a solubility of 94.62, 78.94 and 93.12 mg/mL respectively.

Construction of pseudoternary phase diagrams
A phase diagram was constructed on the basis of the solubility of drug, oil, surfactants, cosurfactants and aqueous phase. The aqueous titration was performed with addition of water (5 % increment of up to 95 % [14]. As shown in Figure 2, the area of nanoemulsion region in the phase diagram appeared to have decreased minimally when the S mix ratio increases. The S mix 1:1 shows a larger nanoemulsion region than that of 3:1.
It was also noticeable that the S mix 1:1 ratio took up a greater amount of water and still remained as a clear mixture during aqueous titration. In Figure 2, the region A forms a clear emulsion while B & C formed more turbid and milky emulsions.

Preparation of nanoemulsion
The nanoemulsifying region incorporating 1 % W/V of 5FU in S mix 1:1 appeared to be larger (p < 0.05) than that of the other S mix formulation ratios (1:2 and 1:3) in the presence of drug ( Figure  2(a)). Six points were randomly picked from the self-nanoemulsifying region from S mix 1:1 and each formulations composition are shown in Table 1.

Droplet size
The mean droplet size of 5-FU nanoemulsions was in the range of 80.24 -112.87 nm. The droplet size of nanoemulsion was increased with the increase in concentration of oil (Capyrol PGMC) as shown in Table 2.    The formulation containing 60 % (FU3) oil has the highest particle size (112.87 ± 1.78) and formulation containing 20 % water and 50 % oil has the lowest particle size (80.24 ± 2.14) nm) as compared to other formulations. The results from Table 2 show that all the formulations had droplets in the nanometer range with low values of polydispersity index.

Thermodynamic stability
The results of stability testing for the nanoemulsion are shown in Table 3. All of the optimized formulations passed the thermodynamic stability testing (freeze thaw cycle) as none of them exhibited any sign of phase separation or drug precipitation at the end of the cycles.

In vitro skin permeation
The in vitro release profiles of 5FU from its various nanoemulsion formulations containing 1 % w/v of 5FU (FU1, FU2, FU3, FU4) and plain carbopol gel containing 1 % of 5FU are presented in Figure 3.
The in vitro skin permeation was found to be highest for formulation FU1 and lowest for FU3 (Figure 3). The skin permeation profile of FU1 was significantly (p < 0.05) higher as compared to FU3. In vitro skin permeation parameters of prepared nanoemulsion formulations and control are shown in Table 4.
It was observed that the permeation parameters cumulative amounts of drug permeated, steadystate flux values, enhancement ratios and permeability coefficients in all nanoemulsion based formulation (FU1, FU2, FU3 and FU4) were found to be significantly (p < 0.05) higher than that of FU loaded conventional gel at the end of the 8h permeation study as shown in Table 4. The amount of drug permeated from its nanoemulsion formulation through the skin can be arranged in the following descending order: FU1>FU3>FU2>FU4, where the amounts of the drug penetrated after the 8h of study were 196.36, 156.25, 136.25 and 112.21 µg/cm 2 , respectively. In contrast, in vitro skin permeation of conventional gel containing 1 % 5FU was observed to be significantly (p < 0.05) lower (60.12 µg/cm 2 ) at the end of 8h.

In vivo skin deposition
Based on the in vitro skin permeation results, the nanoemulsion formulation FU1 and the aqueous cream were selected for in vivo skin deposition studies. The amount of drug retained on mice skin for different time intervals (1h, 2h, 4h and 8h) are shown in Figure 4. It was observed that the there was no significant (p < 0.05) difference in skin deposition in FU2 at different time intervals and that the maximum amount of deposition (292.45 µg/cm 2 ) was reached at the end of 2 h at which point there was no further increase of drug deposition observed. On the other hand, it was noticed that the amount of drug retained in 5FU loaded conventional gel was 121.42 µg/cm 2 at the end of the 8 h study and this was attributed to a lower ability of the cream formulation to penetrate the skin layers. Finally, all the nanoemulsion based formulations (FU1, FU2, FU3 and FU4) showed good in vitro penetration, in vitro skin deposition, and in vivo skin deposition in mice skin. Out of all the formulations, FU1 showed the maximum in vitro penetration and in vivo skin deposition as compared to the other formulations (Figure 3 &  4).

Skin irritation
The study was conducted to test the safety and skin irritation caused by formulations. The mean erythema scores upon application of optimized nanoemulsion FU1, 5 % W/V SLS solution control (placebo) and carbopol plain gel of 1 % 5FU are shown in Figure 5. The SLS solution (5 % W/V) was used as standard irritation in this study. Results indicate that erythema caused by SLS were much higher (4.1 level score) compared to FU1 and plain gel containing 1 % 5FU.

DISCUSSION
The drug has shown the highest solubility in Transcutol HP and it has been chosen as the surfactant for this study. Similarly, PEG 400 and Capyrol PGMC were chosen as the oil and cosurfactant, respectively, for this study. Furthermore, Transcutol HP reported to be very attractive as a penetration enhancer due to its non-toxicity and biocompatibility with the skin [14]. The right blend of oil and surfactants with the right value of hydrophilic lipophilic balance (HLB) leads to the formation of stable nanoemulsion formulations. In this study, HLB value of blend of oils and surfactants were found to be ~ 5.3 which is in the range for W/O type of emulsion.
In Figure 2, diagram (a) has shown the largest emulsification area. This could be explained by the fact that a higher number of surfactants can be absorbed at the interface, thus stabilising the formation of nanoemulsions [15]. Hence, the ratio of 1:1 was selected for formulation development and 6 random points within this area was selected to be used for further studies. A pseudoternary phase diagram in the presence of 1 % W/V 5-FU was also plotted with S mix 1:1 to determine the effect of drug on the phase diagram [16]. S mix 1:1 ratio showed no difference in the area of nanoemulsion whether with the presence or absence of 1 % 5FU.
Determining the droplet size is of great importance in nanoemulsion formulations as small globule sized emulsion contributes to a greater interfacial area which can then provide better drug partitioning and absorption. However, there is no consensus in the literature on the exact size range of a nanoemulsion [16]. The polydispersity values of all formulations were very low which indicates uniformity of droplets within the formulation. The Zeta potential of the formulation was also measured in order to determine the interaction between colloidal particles, where large positive or negative values suggesting high inclination of emulsion repulsion and hence, stability [17].
The results from thermodynamic stability studies of the optimized preparation shows good stability against acceleratory stability condition. The average particle size and physical appearance of the formulation after the stability study at extreme conditions was observed and the results indicated that there were no significant changes in particle size or physical appearance (Table 5). The significant difference in drug permeation between nanoemulsion formulations FU1 and FU3 was probably due to the mean size droplets [18], which were significantly smaller for FU than for FU2 and FU3 and also due to the higher amount of penetration enhancer (Transcutol and Capyrol PGMC) used in the FU1 formulation which may work by interacting with the structural components of the stratum corneum to enhance drug penetration [19].
It was observed that the in vitro skin permeation parameter values of 5FU gel preparation were much lower than that of the nanoemulsion shown in Figure 4. This could be due to the low flux and high viscosity of the formulation which can explain the lesser in ability for the aqueous solution to penetrate skin layers as compared to nanoemulsion formulations as shown in Figure 4. In this study, Transcutol was used as the surfactant in this study and this compound has been reported for its skin penetration-enhancing effect [18,19]. Capyrol PGMC was used as the oil in this study and this oil is known for its penetration-enhancing effect by altering the skin texture [20]. Moreover, the characteristics of nanoemulsions such as high drug concentration, the small droplet diameter and the reaction between the surfactants and skin stratum corneum could have been contributed to the permeation-enhancement of the prepared formulations.
From the above in vivo and in vitro permeation data, it was found that all of the prepared nanoemulsions could improve the skin permeation of 5FU over the conventional gel that has the same drug concentration. This could be, as reported previously, the thermodynamic activity of formulations which can be described as viscosity is important to the permeation into skin. It is known that the viscosity of nanoemulsions is much lower than that of gel, so the mobility of drug in nanoemulsions is more facile. Furthermore, the nanoemulsions may affect the stratum corneum structure and reduce the diffusional barrier by acting as a permeation enhancer [20,21].
In the skin irritation study, the mean irritation score of FU1 was significantly lesser than that of the plain gel. The results clearly indicate that the optimized nanoemulsion formulation was well tolerated by rat skin and produced no irritation to skin. Based on this, it seems that the optimized nanoemulsion formulation may be safe used for topical drug delivery compared to conventional gel or cream formulations of the drug.

CONCLUSION
The penetration of 5FU into the skin and its retention within the skin is desirable for the treatment of many skin diseases, including skin cancer. Thus, the developed nanoemulsion formulation has great potential for topical delivery of 5FU for safe and more effective treatments of various skin diseases including skin cancer. However, further studies, such as extensive pharmacokinetic, histopathological and toxicity studies are required to fully develop these formulations for therapeutic application.