extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Chemistry Biology

text:
Measuring the dynamic surface accessibility of RNA with the small paramagnetic molecule TEMPOL

The surface accessibility of macromolecules plays a key role in modulating molecular recognition events. RNA is a complex and dynamic molecule involved in many aspects of gene expression. However, there are few experimental methods available to measure the accessible surface of RNA. Here, we investigate the accessible surface of RNA using NMR and the small paramagnetic molecule TEMPOL. We investigated two RNAs with known structures, one that is extremely stable and one that is dynamic. For helical regions, the TEMPOL probing data correlate well with the predicted RNA surface, and the method is able to distinguish subtle variations in atom depths, such as the relative accessibility of pyrimidine versus purine aromatic carbon atoms. Dynamic motions are also detected by TEMPOL probing, and the method accurately reports a previously characterized pH-dependent conformational transition involving formation of a protonated C–A pair and base flipping. Some loop regions are observed to exhibit anomalously high accessibility, reflective of motions that are not evident within the ensemble of NMR structures. We conclude that TEMPOL probing can provide valuable insights into the surface accessibility and dynamics of RNA, and can also be used as an independent means of validating RNA structure and dynamics in solution.

INTRODUCTION
Surface accessibility plays a key role in delineating molecular interactions. However, accessible surface areas, calculated on the basis of static 3D structures, may not accurately describe the effective atom exposure to the bulk solution. In fact, dynamic features such as local flexibility (1,2), and/or hydration shell motions (3)(4)(5), have been observed to modulate surface accessibility in proteins.
Experimental studies on protein dynamics are, indeed, important in structural biology, and complement the otherwise 'static' view of molecular structure. In this respect, soluble paramagnetic probes have been shown to be useful in obtaining insight into processes regulating protein surface accessibility. In multidimensional NMR spectra, soluble paramagnetic molecules cause distancedependent paramagnetic relaxation enhancement (PRE), manifested as line broadening and attenuation of crosspeak intensity. The extent of attenuation depends upon the local concentration of the paramagnetic probe. Thus, higher atom exposures result in higher degrees of paramagnetic attenuation (6,7). Recently, PRE studies have been widely applied in the investigation of protein-ligand (3), protein-protein (8)(9)(10)(11) and protein-DNA (12) interactions, as well as in the localization of protein active sites (1,3,5,6,13,14). PRE studies are capable of efficiently detecting transient intermediates and short-lived encounter complexes that may otherwise go undetected by other methods (11,12).
The absence of strong interactions between biomolecules and paramagnetic probes is a prerequisite for probing surface accessibility. In the past few years, four paramagnetic agents with different sizes and chemical characteristics have been selected to fulfill this requirement: TEMPOL (3,5,6,7,15), Gd(III)DTPA-BMA (16), Gd 2 (L7)(H 2 O) 2 (10) and O 2 (2). Among these probes, TEMPOL has been used most extensively in protein accessibility studies, and a large amount of data on the mechanisms modulating the TEMPOL approach on protein surfaces is available (3,5,7,10,15,16). TEMPOL is soluble and small, with a radius of 3.7 Å ( Figure 1A). Thus, TEMPOL is small enough to enter the narrow major groove of an RNA helix, and is therefore an ideal probe for investigating the surface accessibility of RNA. The paramagnetism of TEMPOL is due to the unpaired electron on the nitroxide group ( Figure 1A), which induces fast relaxation of nuclear spins through electron-nuclear dipole-dipole coupling. In the absence of specific interactions, TEMPOL approaches a molecular surface randomly, and thus the paramagnetic nitroxide group can be considered to be at the center of a sphere with a radius of 3.7 Å (the radius of TEMPOL). The paramagnetic effect of TEMPOL is exerted in a distance dependent, through-space manner, with >90% of relaxation induced through distances of 10 Å or less from the paramagnetic center (7).
Here, we show that TEMPOL induced PRE is a facile method that can be used to investigate the surface accessibility of RNA. For stable helical regions of RNA, there is excellent agreement between the surface accessibility and the position of the atom within the structure. Furthermore, we show that the method is capable of detecting dynamic conformational transitions. Some dynamic regions result in higher levels of PRE than would be predicted from the corresponding ensemble of NMR structures, suggesting that the method detects transient conformational dynamics that are 'invisible' with respect to the NMR-derived structures. Therefore, TEMPOL probing provides a facile way to independently validate and complement RNA structure models and dynamic measurements.

Sample preparation and NMR measurements
The investigated RNA's, whose sequences are shown in Figure 1, were prepared by in vitro transcription as previously described (17,18). All NMR samples were $1 mM in D 2 O. The pH was adjusted to 6.8 for the HIV-1 RNA, and 8.0 and 5.7 for the U6 ISL RNA NMR samples. Paramagnetic samples contained an optimal 20 mM TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-1 oxyl, 97% purity, Sigma-Aldrich) concentration, which was achieved by adding directly to the NMR tube a few microliters of a 2 M TEMPOL solution. Stock TEMPOL solutions were prepared by directly dissolving TEMPOL into 99.9% D 2 O to a final concentration 2 M.
Since denaturing effects of TEMPOL on RNA structures could not be excluded a priori, after each addition of a 2 M solution of TEMPOL to the $1 mM RNA solutions, 2D TOCSY spectra were recorded to check for chemical shift differences due to TEMPOL. No significant chemical shift changes upon TEMPOL addition were observed.
Cross-peak volumes were measured with a greater than 90% confidence level using the Sparky integration tool (http://www.cgl.ucsf.edu/home/sparky).

Cross-peak attenuations
The cross-peak volumes were autoscaled according to the relation (13): where i p,d is the autoscaled volume of peak i from the paramagnetic or diamagnetic spectra, V i p,d is the measured peak volume from the paramagnetic or diamagnetic spectra, and n is the number of peaks measured. Hence the autoscaled volume is equal to the measured volume divided by a scaling factor corresponding to the mean cross-peak volume over n molecular locations. Paramagnetic attenuations, A i 's, were calculated from the autoscaled diamagnetic and paramagnetic peak volumes, respectively, d and p , according to the relation (13):

Depth index calculation
The 3D atom depths, reported as depth indexes D i,r 's, were calculated on the basis of the NMR solution structures (PDB codes: 1PJY, 1SY4, 1SY7) (17,20,21) using the SADIC software (22). For each RNA, reported D i,r 's were averaged over the whole NMR structure ensemble. D i,r is defined according to the relation (22): where V i,r is the exposed volume of a sphere of radius r (sampling radius) centered on atom i and V 0,r is the total volume of the same sphere. The sampling radius was set to 10 Å , which corresponds to the practical limit of the paramagnetic effect (7). The probe radius, used for the computation of the macromolecular surface, was set to 3.7 Å corresponding to the TEMPOL molecular radius as calculated by using the MOLMOL software (23). For each C-H correlation, the entire ensemble of 20 NMR structures were analyzed and the averaged D i,10 is reported. For simplicity, the averaged D i,10 value is referred to in the text as D i .

Molecular dynamics
Several MD simulations were performed in explicit solvent starting from the lowest energy solution structures of the investigated RNA's (PDB codes: 1PJY, 1SY4) (17,21,24) by using the AMBER package (25) and the AMBER-99 force field (26). Each RNA was centered in a truncated octahedron and the initial shortest distance between the RNA atoms and the box boundaries was set to 10 Å . Then, an optimal amount of sodium ions was added in order to generate a neutral system. The remaining box volume was filled with TIP3P type water molecules (27). Water and ions positions were minimized with 500 steps of steepest descent plus 500 steps of conjugate gradient by keeping the RNA fixed. Then, the entire system was energy minimized with 1500 steps of steepest descent followed by 1000 steps of conjugate gradient minimization. Then, each system was first equilibrated in a 20 ps run where temperature was gradually raised from 0 to 300 K. Hence, the equilibrated systems were simulated by keeping the temperature (300 K) and pressure (1 atm) constant. Thus, a weak coupling to external pressure baths was applied (relaxation time 1.0 ps) (28), while Langevin thermostat was used to control the temperature (collision frequency 1.0 ps À1 ) (29). Bonds were constrained by SAHKE algorithm (30). Non-bonded interactions were accounted by using the PME method (31). An integration time step of 2 fs was used and trajectories were saved every 0.2 ps.
The backbone root mean square deviation (r.m.s.d.) of investigated RNA's over the MD runs ( Figure S1, Supplementary Data) level off after equilibration periods shorter than 500 ps in all the cases. Thus all the subsequent analyses of the trajectories were carried out from 500 ps onward.

The stable HIV-1 frameshift site stem-loop
The frameshift site stem-loop RNA of HIV-1 is a highly conserved and very thermodynamically stable (T M > 908C) RNA domain ( Figure 1B) (17,19,32). Due to its high stability and regular shape, it is a good initial model RNA to investigate the use of TEMPOL as a probe of RNA surface accessibility.
2D 1 H-13 C HSQC NMR spectroscopy is particularly suited for probing the macromolecular surface with TEMPOL, since the paramagnetic attenuation affects cross-peak intensities arising from covalently bound 13 C and 1 H nuclei that are very close in space (3). Furthermore, due to a favorable chemical shift dispersion, the aromatic CH correlations are suitable HSQC signals for delineating the accessibility of RNA structures. The TEMPOL generated PRE effects were analyzed for all the aromatic C2-H2, C6-H6 and C8-H8 correlations of the HIV-1 frameshift site RNA. The PRE values were quantitated as attenuation index (A i ) values, as described in the Materials and Methods section.
The TEMPOL induced A i values were mapped to the surface of the RNA, and also plotted graphically (Figure 2A and B). The data correlate well with the structure of the HIV-1 RNA. The tetraloop and terminal ends of the RNA are more accessible than the A-form helical region, suggesting that 3D structure is the main factor modulating the surface accessibility of the RNA. Intriguingly, we noticed that the purine aromatic C8-H8 correlations have overall higher A i values than the pyrimidine aromatic C6-H6 correlations. Quantitation of atom exposure, expressed as 3D atom depth index (D i ) (22) values, confirmed that the purine C8-H8 groups are more exposed than the pyrimidine C6-H6 groups by an average value of 8% over the interior of the helix (residues 2-8 and [15][16][17][18][19][20][21]. The agreement between the average atom depth (D i ) calculated from the ensemble of NMR structures and attenuation index (A i ) is shown in Figure 2C. In general, CH groups having high D i values, i.e. are close to the molecular surface, exhibit high A i . In contrast, CH groups more buried in the structure have low or intermediate A i values. This trend holds true even for nucleotides that have minor differences in atom exposure. For example, the first 5 0 nucleotide is more accessible than the last 3 0 nucleotide, even though they both reside at the helical end ( Figure 2B). This is because the 5 0 aromatic carbon (1C8, D i = 0.786) is indeed more exposed than the 3 0 aromatic carbon (22C6, D i = 0.491), as indicated by its higher D i value ( Figure 2C). This is in agreement with chemical acylation studies showing that RNA helical 5 0 ends are more accessible than 3 0 ends (33). However, anomalously high A i 's are observed for nucleotides in the tetraloop region (A10, A12, A13 and G14), which is a less ordered region of the structure (17). In particular, the C8 groups of A12 and A13, corresponding to the 3 0 side of the tetraloop, are highly attenuated, even though they are well stacked and relatively buried within the ensemble of NMR structures. These data suggest that the conformational variation within the time averaged NMR ensemble does not reflect the true conformational dynamics of the molecule. The NMR structures of RNA loop regions are largely defined by NOE distance restraints ( 5 Å ), and the unstacking of loop nucleotides may go undetected by NOE methods, since such transitions may result in internucleotide distances >5 Å . The paramagnetic attenuation of the tetraloop nucleotides suggests a strong connection between accessibility and RNA conformational flexibility.
To further investigate the accessibility-flexibility correlation, we performed a 10 ns MD simulation on the NMR structure of the HIV-1 frameshift site RNA (PDB code: 1PJY) (17). As expected, the root mean square fluctuations (r.m.s.f.) plot shows enhanced flexibility for the tetraloop and terminal nucleotides (Figure 3). In contrast, the helical residues experience limited and homogeneous fluctuations, consistent with the high stability previously observed for this RNA stem (19). By comparing the attenuation and the r.m.s.f. data ( Figures 2B and 3), the correlation between accessibility and structural fluctuations is apparent.

The dynamic U6 intramolecular stem-loop
The U6 intramolecular stem-loop (ISL) is a highly conserved domain of the small nuclear U6 RNA ( Figure 1C), and is involved in pre-messenger RNA splicing (34). The NMR solution structure determined at pH 7.0 (PDB code: 1SY4) (18,21) shows that the U6 ISL is comprised of two A-form helices separated by an internal loop containing a partially protonated C67-A79 wobble base pair and an unpaired U80 that is predominately stacked in the helix. The helix is capped by a GCAUA pentaloop that contains a sheared G71-A75 base pair and a 3 0 adenine stack, facilitated by the bulged nucleotide U74. Hence, this RNA alternates between structured and dynamic regions, enabling us to further investigate the relationship between accessibility and structural flexibility. Analyses of NMR line shapes and relaxation rates indicated that the U6 ISL RNA displays motions on both the ps-ns and ms-ms timescales (20,35). Furthermore, the U6 ISL undergoes a pH-dependent conformational switch induced by protonation of A79 (pKa = 6.5), which results in stabilization of the C67-A79 wobble pair and base flipping of the neighboring U80 nucleotide (20,21,35). The C67-A79 wobble pair has a lifetime of 20 ms, and the base flipping of U80 occurs on an $80 ms timescale (20,35). Since the dynamics of this RNA have been previously characterized by NMR, it is an ideal model system for exploring the application of the TEMPOL PRE analysis in detecting RNA conformational transitions.
Since the pH-dependent conformational transition within the U6 ISL is directly correlated to the protonation state of A79 (35), the accessibility studies were performed at pH 8.0 and at pH 5.7. These experimental conditions assure minimal interference from the other conformer (population < 10%) (20). The TEMPOL PRE analysis was possible for 26/30 and 25/30 aromatic CH correlations, respectively, for the RNA at pH 8.0 and pH 5.7. The remaining resonances could not be reliably analyzed due to chemical shift overlap. The pH 8.0 attenuation profile ( Figure 4A and B) correlates well with the U6 RNA structure. Higher A i values are observed for the terminal nucleotides (62 and 85), the pentaloop and adjacent nucleotides (72-76; 71 was not measured due to spectral overlap), and the internal loop nucleotides (79 and 80).
Inspection of Figure 4C shows the good agreement between accessibility and atom exposure. All of the anomalously high A i values are associated with nucleotides located in the dynamic pentaloop and internal loop regions (residues 73, 75, 76 and 79). The greatest anomaly is found for the A79 C2-H2 correlation, which experiences the highest TEMPOL accessibility at pH 8.0, in spite of its relatively low exposure in the pH 7.0 NMR solution structure. The A79 C2 atom is directly adjacent to the partially protonated nitrogen (A79 N1). Interestingly, the dynamics of protonation (20), as well as the relaxation rates for the base (35) suggest that A79 is indeed dynamic and must be accessible to solvent when unprotonated, although the solvent-accessible conformation could not be directly observed in the time-averaged ensemble of NMR structures calculated at pH 7.0 (20,35).
The conformational switch induced by lowering the pH to 5.7 caused changes in the TEMPOL PRE pattern which are mainly located in the internal loop region ( Figure 5A and B). In particular, the most significant change is the large decrease in A i (1.1-0.5) detected for A79 C8. This is in agreement with the structure and thermodynamics at pH 5.7 (20), which indicate that the internal loop structure is stabilized by formation of a protonated C67-A79 wobble pair at low pH, and that this base pair is fully stacked within the helix. Furthermore, an increase in A i (1.1-1.4) is detected for U80 C6. The A i value of 1.4 is consistent with an increase in the population of the baseflipped conformation for U80. As a comparison, the extrahelical U74 nucleotide experiences dynamics on the ps-ns timescale in a manner that is not pH-dependent (35) and has an A i value that does not change with pH ( Figure 5B). Additionally, the A i values for the extrahelical nucleotides U74 and U80 are very similar at pH 5.7, and due to their extrahelical conformations, both nucleotides are also predicted to have high D i values ( Figure 5C). Although the increase in A i for U80 correlates with an increased proportion of the base-flipped conformer at low pH, as expected (20), the increase in A i for U80 relative to the pH 8.0 sample is less than might be expected, since the difference between the two measurements (0.3 A i units) is barely above the observed standard deviation ( Figure 5B). This is likely due to the fact that the pH 8.0 A i value of 1.0 is relatively high, which may be reflective of the fact that a 3% population of based-flipped conformer still exists at this pH (35). Relative to the 80 microsecond base-flipping timescale (35), TEMPOL probing is very fast and results in irreversible PRE, allowing detection of even very transient populations. Therefore, the relatively small difference in A i values ( Figure 5B) for U80 is likely to be due to the detection of the base-flipped conformer even when it is present as a minor poplulation. On the other hand, the low pH condition locks A79 into a protonated wobble base pair with C67, which results in a more pronounced difference in attenuation for the A79 C2 and C8 resonances ( Figure 5B). The flipped out U80 nucleotide may help shield A79 from TEMPOL, which could further account for the large drop in attenuation for the A79 C8-H8 correlation.
The A79 C2 resonance has an anomalously high A i value at pH 8.0 ( Figure 4C), suggesting that it is much more exposed than its position in the NMR structure, which was previously determined at pH 7.0 (21). Furthermore, interpretation of NMR relaxation rates at this site are complicated by chemical exchange due to the rapid protonation equilibrium of the adjacent N1 group (20,35). Therefore, in order to gain a better understanding of the dynamic motions associated with the unprotonated A79 conformation, an 18 ns MD simulation was performed starting from the pH 7.0 solution structure (PDB code 1SY4) (21) using the condition described in the Materials and Methods section. A low stability for the simulated trajectory can be observed by analyzing the RMSD versus time plot ( Figure S1, Supplementary Data). In fact, during the MD trajectory, a switch from an unprotonated, single hydrogen bond C67-A79 wobble pair to a single hydrogen bond C67-U80 pair (C67 amino to U80 O4) is observed at the beginning of the simulation. This conformation was stable for $3.5 ns, then underwent a base-pairing opening event for $3 ns and subsequently reached a new base-pair conformation containing a double hydrogen-bonded C67-U80 pair (C67 amino-U80 O4, C67 N3-U80 imino) that was stable until the end of the simulation (Figure 6). This variation in the internal loop structure left A79 unpaired but still stacked in the helix. However, the MD sampling was not able to reach a stable orientation for this nucleotide that sampled many alternative conformations during the last 10 ns of trajectory. This behavior was also observed for an 11 ns MD simulation on a longer U6 ISL sequence (PDB code: 1XHP) (36), which has given similar results ( Figure 6 and Figure S1, Supplementary Data). In order to relax the internal loop structure in the best local minimum, 10 conformations were extracted at regular intervals from the last 10 ns of simulation. Subsequently these structures were energy minimized using the simulated annealing protocol summarized in Figure S2, Supplementary Data. A comparison of the lowest energy MD-derived internal loop conformation and the lowest energy NMR solution structure is shown (Figure 7). In contrast to the pH 7.0 NMR structure, the unprotonated (high pH) internal loop forms a base triple in which C67 is no longer planar and A79 is rotated $258 toward the minor grove. This conformation effectively increases the surface exposure of the A79 C2 group in a manner that is more consistent with the TEMPOL attenuation data (D NMR

RNA surface accessibility
Investigating how soluble paramagnetic probes approach protein systems has been shown to provide useful information on the dynamic processes occurring at the protein-solution interface (1)(2)(3)(5)(6)(7)(8)(9)(10)14). Here, the surface accessibility of RNA molecules characterized by different shape and dynamics was analyzed by evaluating the PRE generated by TEMPOL on conventional 2D NMR measurements. The present results show that TEMPOL approaches RNA surfaces more uniformly than proteins (1,3,6,10,13,14). In fact, the agreement between the paramagnetic attenuation and atom depths for helical RNA molecules ( Figures 2C, 4C and 5C), suggests that the shape, local dynamics and composition of RNA molecular surfaces are more homogeneous than those of proteins. Furthermore, the high negative charge typical of RNA surfaces may hamper formation of transient aggregates, which have been shown to significantly modulate the surface accessibility of proteins (5,(8)(9)(10). However, we find that nucleotides residing in flexible loop regions show higher accessibility than the ones observed for helical residues of similar depth (Figures 2-5). This finding suggests that these nucleotides transiently unstack, increasing the exposure of the base to the bulk solvent. In many cases, such transient events may not be evident within NMR structures that are typically time-averaged and population-weighted ensembles. Recently, the transient unstacking of stable GNRA tetraloops has been observed using femptosecond timescale fluorescence spectroscopy (37).
Detecting RNA conformational changes RNA conformational transitions are essential for ribosome and spliceosome function and play a significant role in recognition of proteins, RNA and ligands (38)(39)(40)(41). Thus, studying RNA conformational dynamics is of primary relevance in understanding the correlation between RNA structure and function. However, investigating conformational changes in solution may be a complex and time-consuming endeavor. Hence, alternative methods able to map conformational transitions in RNA may be very helpful in complementing other investigation techniques. In the present report, we show that a small paramagnetic probe can detect a pH-dependent conformational switch in U6 RNA that has implications for spliceosome function and catalysis (20,35). The detailed pathway and associated energetics of this conformational change are unknown, but are driven by protonation of A79 (20,35). Here, we observe formation of the protonated C67-A79 pair causes large A i variations in the internal loop region ( Figure 5B). Comparison of the TEMPOL probing data (Figures 4  and 5) support the hypothesis that the transition between the protonated and unprotonated U6 ISL proceeds through an intermediate structure that is protonated but has a more exposed A79 (35). Interestingly, our MD simulation suggests a new conformation for the unprotonated U6 ISL internal bulge, in which the A79 base is rotated toward the minor grove, enhancing its solvent exposure (Figure 7). The MD simulation suggests that U80 is at least transiently involved in a base triple with C67 and A79. This may help explain the preference for this nucleotide to adopt a stacked conformation at high pH.
Further A i variations are observed for nucleotides flanking the U6 ISL internal loop (G77 and G78) that show enhanced TEMPOL accessibility at low pH. These nucleotides are in stable Watson-Crick pairs, and previous thermodynamic investigations indicate that the structure is even more stable at low pH (21), dismissing the possibility of increased conformational flexibility for these nucleotides. Possibly, the modified molecular shape as well as the proximity of the highly dynamic loop nucleotides could increase the self-diffusion properties of the hydration layer surrounding these nucleotides, thus enhancing their accessibility to the paramagnetic probe (3)(4)(5)42).
Recently, we have demonstrated that TEMPOL probing can be used to detect the binding of a small molecule ligand to the surface of the HIV RNA (43). Therefore, the TEMPOL probing method is well suited for probing RNA structure, dynamics and ligand interactions.

SUPPLEMENTARY DATA
Supplementary Data are available at NAR Online.

ACKNOWLEDGEMENTS
This study made use of the National Magnetic Resonance Facility at Madison (www.nmrfam.wisc.edu), which is supported by National Institutes of Health grants P41RR02301 (Biomedical Research Technology Program, National Center for Research Resources) and P41GM66326 (National Institute of General Medical Sciences). Equipment in the facility was purchased with


extfieldsofstudy: Chemistry Biology

text:
Free Energy of Activation for the Comorosan Effect

Initial reaction rate data for lactic dehydrogenase / pyruvate, lactic dehydrogenase / lactate and malic dehydrogenase / malate enzyme reactions were analyzed to obtain activation free energy changes of -329, -195 and -221 cal/mole, respectively, for rate increases associated with time-specific irradiation of the crystalline substrates prior to dissolution and incorporation in the reaction solutions. These energies, presumably, correspond to conformational or vibrational changes in the reactants or the activated complex. For the lactic dehydrogenase / pyruvate reaction, it is estimated that on the order of 10% of the irradiation energy (546 nm, 400 footcandles for 5 seconds) would be required to produce the observed reaction rate increase if a presumed photoproduct is consumed stoichiometrically with the pyruvate substrate. These findings are consistent with the proposition that the observed reaction rate enhancement involves photoproducts derived from oscillatory atmospheric gas reactions at the crystalline enzyme substrate surfaces rather than photo-excitations of the substrate molecules, per se.

Introduction
Transition-state theory and its associated free energy of activation serve as the primary tools in conceptualization of models for enzyme catalysis mechanisms. These models are becoming increasingly elaborate, and hypothetical, as they struggle to account for the enzyme's specificity and efficiency (e.g., see Ma et al., [1]). Modeling necessarily is based on the assumption that all relevant features of the catalytic process are taken into consideration. With regard to this point, a body of experimental work referred to as the Comorosan effect may prove relevant. Here, we present an evaluation of some implications of that work from a transition-state theory perspective.
The Comorosan effect is a phenomenon in which the initial velocity of an enzymatic reaction is increased as a consequence of utilizing substrate that had been irradiated in the crystalline state, for a specific time duration, prior to dissolution and incorporation in the reaction mixture. This behavior has been observed for the reactions of over twenty enzymes isolated from multiple sources (see Table 1) and, thus, may reflect a very common, perhaps even fundamental, property of enzyme catalysis. To date, it has not been established how the relevant irradiation energy is absorbed by the crystalline material, how it is transformed on dissolution, nor how it is manifest in producing an enhanced in vitro enzymatic reaction rate. No assessment of the energetics attendant to the observed reaction rate stimulation has been reported. For overviews of much of the published work in this area, see Comorosan et al , [2,3].
Comorosan [4,5] sought to explain the phenomenon as due to photo-excitation of the irradiated crystalline enzyme substrate molecules, per se, to special "biological observable" quantum states detectable only with the extreme sensitivity of enzymes. The purpose of this theoretical investigation is to derive an estimate of the magnitude of the energy that is involved, particularly with respect to Comorosan's model. We applied the transition-state theory of reaction rates to kinetic data published by Comorosan and coworkers for three enzyme reactions, the lactic dehydrogenase interconversions of pyruvate and lactate [6] and the malic dehydrogenase conversion of malate to αoxaloacetate [7].
In the simplest representation of transition-state theory [8] for an enzymatic reaction, one has: where k 1 is the rate constant describing formation of the activated complex and k 2 that for its conversion to product. Formation of the transition-state complex requires a free energy of activation, ΔG*, usually illustrated as a barrier along the reaction coordinate.
The basic modeling assumption made here is that the energy absorbed and transferred from the irradiated crystals, in whatever form, serves to reduce ΔG*, thereby increasing the reaction rate. In general, this might be achieved either by raising the free energy level of the free reactants or of the Michaelis-Menten complex (if considered distinct from the transition-state complex), or by lowering that of the transition-state complex.
The rate, v, of the reaction can be expressed as: v = [Complex] x (rate of traversing the energy barrier) .
The rate of traversing the energy barrier is given by κK B T/h , where κ is a transmission coefficient giving the probability that formation of the complex will lead to reaction, K B is Boltzmann's constant, T is the absolute temperature and h is Planck's constant. The transmission coefficient usually is assumed to be unity or very nearly so, and will be taken as such here. Thus, The equilibrium constant for the complex, K*, is given by:  Of interest here is the limiting (maximal) rate of reaction, that is, the initial reaction rate, achieved when all reactants are present in excess relative to the enzyme. This is the effective rate at an instant after t = 0 and before a significant portion of any reactant can be removed or product accumulated.

Methods
In modeling the experimental data at hand, we assumed that irradiation of the crystalline substrate creates an entity, or precursor thereof, which though unidentified, we shall designate as Є. Inspection of the published reaction rate curves strongly suggest that it is consumed in the course of the reaction, the presumed product being here designated Є'.
This entity may, or may not, be identifiable with excited molecules of the substrate. In the subsequent enzyme assay, two simultaneous reactions can proceed: (1) S + E S:E P + E and (2) Є + S + E Є:S:E P + E + Є' (for notational simplicity, the cofactor, NADH or NAD + , is not represented).
Here, two competing models may be envisioned. If Є corresponds simply to an excited state of the substrate, as proposed by Comorosan, then the activation energy barrier for the catalyzed reaction will be reduced as a consequence of increased initial energy of the substrate. In this case, one would anticipate that the concentration of Є must be some appreciable fraction of that of the substrate. On the other hand, Є may correspond to an altogether different chemical species that interacts with the enzyme to alter its conformation in a manner that increases its catalytic potency, thus lowering the peak of the activation energy barrier. In this alternate case, the concentration of Є would need be only a fraction of the concentration of the enzyme, typically orders of magnitude less than that of the substrate.
Under the conditions of the experiments being examined, concentration of the reactants, other than Є, are sufficiently high to be treated as constant. For the control reaction (non-irradiated substrate), the measured reaction rate is zero-order throughout with respect to both substrate and cofactor (e.g., pyruvate and NADH). Its rate constant is designated k 0 . For the reaction involving irradiated substrate, the initial reaction rate is faster and then decreases (presumed due to consumption or degradation of Є ) to the same constant rate observed for the control reaction. Its initial rate constant at t=0 is designated k ε .
Data points for NADH absorbance versus time were estimated visually from the published graphs for the three reactions by photo enlargement onto a grid (estimation error <2%). The two data sets for each reaction with and without prior irradiation (absorbance designated A ε and A 0 , respectively) were utilized to determine k 0 and k ε by simultaneously fitting them to the following differential equations using nonlinear regression analysis: LDH/Pyruvate reaction: LDH/Lactate and MDH/Malate reaction: Curve fit values for k ε and k 0 were converted from absorbance units / sec to moles NADH / sec noting that for the LDH / pyruvate reaction, the initial absorbance was 0.480 units for the solution containing 0.34 μmoles of NADH.
All calculations were performed using the software package Scientist V.2.01, MicroMath Inc., Salt Lake City, UT.

Results
The fitted curves for the three reactions are presented in Fig. 1, 2 and 3. The experimental parameters and calculated reductions in activation free energies, ΔΔG*, are presented in Table 2.
The LDH / pyruvate data were analyzed further to provide an estimate of the minimum amount of relevant energy that would have had to be absorbed in the irradiation step, assuming that Є corresponds to photo-excited substrate molecules. The 3 mL reaction mixture contained 2.2 x 10 -6 moles of pyruvate and 0.025 x 10 -6 g of LDH, estimated to correspond to 1.8 x 10 -11 moles of LDH (using MW = 140,000 daltons [9]). Inspection of the absorbance versus time data for this reaction revealed that the irradiation effect was associated with a decreased absorbance of 0.030 units that occurs entirely within the first 20 sec of the reaction. Assuming Є corresponds to photo-activated pyruvate molecules and that 100% of these were converted to lactate, then 2. No direct measurements of the amount of irradiation actually absorbed by the crystals have been reported. In the two reports from which the kinetics data utilized here were taken, the irradiation intensity was characterized as being 600 lux. However, in a later publication noting more detailed study of this aspect, Comorosan indicated that the required irradiation intensity, the illuminance, should be approximately 400 footcandles [2], corresponding to on the order of 4000 lux. Using this latter figure (assumed more reliable) along with an estimate of 1 cm 2 for the area covered by the sodium pyruvate crystals and an exposure time of 5 seconds, the total energy to which the crystals were exposed is calculated to be approximately 7.4 x 10 -4 cal. This then would imply that approximately 10% of the total incident radiation must be captured such as to produce photo-excited substrate molecules.

Discussion
On the face of it, one would not expect the crystal irradiation procedure employed in the studies addressed here to have any measurable impact on a subsequent enzyme reaction rate. However, Comorosan and co-workers have published 15 primarily experimental reports involving over 20 different enzymes employing a wide range of reaction rate determination methodologies all of which display this behavior [10,11,12,13]. In addition, 4 collaborative studies [14,15,16,17] conducted in other laboratories and 3 independent studies [18,19,20] have been reported. Some of these include single and double-blind procedures to mitigate against the possibility of experimenter and procedural bias. While a trivial explanation may underlie the phenomenon, such has not been uncovered to date. The particular data examined here for LDH / pyruvate are consistent with other of the above cited reports for that particular reaction, as well as the body of work as a whole.
Early explanations for the phenomenon assumed that the irradiation procedure placed the substrate molecule (e.g., pyruvate, malate, etc.) in an excited state that could be detected (discerned) only by the extreme sensitivity of its enzyme. This model would imply that the photo-activated entity, here designated Є, is, in fact, a sub-population of the substrate and would be consumed stoichiometrically in the conversion of substrate to product.
However, it is estimated above that at least 10% of the irradiation incident on the crystalline material would need be absorbed to provide the required activation energy reduction. This seems highly unlikely, and possibly suggestive of a mysterious lightmatter-biological interaction that lies completely outside contemporary scientific paradigms. More recently, an alternate model has been proposed [21] wherein the crystal irradiation process induces oscillatory free radical mediated reactions involving atmospheric gases at the surface of the crystals. This putative photo-driven process would be similar to a number of observed temperature-driven oscillatory systems involving atmospheric gases [22,23,24]. On cessation of irradiation, defining the t* period, much slower dark reactions would lead to relatively stable, water soluble chemical species which, in turn, are capable of altering reactivity of a particular enzyme.
Thus, a much smaller quantity of the Є species would be required, perhaps only some fraction of the enzyme molecular concentration rather than the orders of magnitude higher substrate concentration (here, for the LDH reaction, 1.8 x 10 -11 vs 2.2 x 10 -6 moles/3 mL).. This behavior would be similar to that observed for the well known action of nitric oxide, an atmospheric gas photo-product, which can induce over a 40-fold increase in the rate of conversion of GTP to cAMP by guanylate cyclase [25,26]. The body of work on this phenomenon reveals that different enzyme reactions may be enhanced by different crystal irradiation time periods (e.g., 15 s rather than 5 s exposure).
Accordingly, it should be anticipated that a small set of relevant chemical species are generated in the photochemical and follow-on dark reactions.

Should this model be proved correct and if the phenomenon indeed reflects a universal
property of enzymes, a new door might be opened to link known physico-chemical processes to pre-biotic evolution and the generalized non-linear dynamics espoused by others [27,28] as fundamental to life processes. Moreover, at some level of refinement, transition state modeling of enzyme reactivity will need to take such a ubiquitous property into consideration. -221 b Temperature = 299 K for each reaction. k 0 = initial reaction rate constant obtained using non-irradiated substrate, moles NADH / sec. k ε = initial reaction rate constant obtained using irradiated substrate, moles NADH / sec. t* = duration of crystalline substrate irradiation at 546 nm. ΔΔG* = -594 ln(k ε / k 0 ) cal/mole.


extfieldsofstudy:
Chemistry
Computer Science
Medicine

text:
Diaquabis(4-methylbenzoato-κO)zinc(II)

The Zn atom in the title mononuclear complex, [Zn(C8H7O2)2(H2O)2], lies on a special position of site symmetry 2. The carboxylate group binds in a monodentate manner so that the geometry is best described as tetrahedral. Adjacent molecules are linked by O—H⋯O hydrogen bonds into a three-dimensional network.

The Zn atom in the title mononuclear complex, [Zn(C 8 H 7 O 2 ) 2 (H 2 O) 2 ], lies on a special position of site symmetry 2. The carboxylate group binds in a monodentate manner so that the geometry is best described as tetrahedral. Adjacent molecules are linked by O-HÁ Á ÁO hydrogen bonds into a three-dimensional network.

Related literature
For related literature, see: Song et al. (2007).

S1. Comment
In the structural investigation of 4-methylbenzate complexes, it has been found that the 4-methylbenzoic acid functions as a multidentate ligand [(Song et al. (2007)], with versatile binding and coordination modes. In this paper, we report the crystal structure of the title compound, (I), a new Zn complex obtained by the reaction of 4-methylbenzoic acid with zinc chloride in alkaline aqueous solution.
As illustrated in Figure 1, the Zn II atom, possesses crystallogarphically imposed C 2 symmetry, which is coordinated by two O atoms from two 4-methylbenzate ligands and two water molecules, and displays a tetrahedral geometry.
Intermolecular O-H···O hydrogen bonding interactions (Table 1) between the coordinated water molecules and the carboxyl O atoms of 4-methylbenzate ligands stabilize the structural packing (Fig. 2).

S2. Experimental
A mixture of zinc nitrate(1 mmol), 4-methylbenzoic acid (1 mmol) NaOH (1.5 mmol) and H 2 O (12 ml) was placed in a 23 ml Teflon reactor, which was heated to 433 K for three days and then cooled to room temperature at a rate of 10 K h -1 .
The crystals obtained were washed with water and dryed in air.

S3. Refinement
Carbon-bound H atoms were placed at calculated positions and were treated as riding on the parent C atoms with C-H = 0.93 -0.97 Å, and with U iso (H) = 1.2 U eq (C). Water H atoms were tentatively located in difference Fourier maps and were refined with distance restraints of O-H = 0.82 Å and H···H = 1.29 Å, each within a standard deviation of 0.01 Å, and with

Figure 1
The structure of (I), showing the atomic numbering scheme. Non-H atoms are shown with 30% probability displacement ellipsoids. Unlabeled atoms are related to the labelled atoms by the symmetry operator (-x, y, 0.5 -z). Refinement. Refinement of F 2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F 2 , conventional R-factors R are based on F, with F set to zero for negative F 2 . The threshold expression of F 2 > σ(F 2 ) is used only for calculating R-factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F 2 are statistically about twice as large as those based on F, and R-factors based on ALL data will be even larger.


Effect of Noble Element Alloying on Passivity and Passivity Breakdown of Ni

Financial support of the Strategic Environmental Research and Development Program (SERDP) through project WP-1415 is greatly appreciated.

During welding of stainless steels (SS), the evaporation and oxidation of Cr from the molten weld pool can result in the generation of carcinogenic hexavalent chromium (Cr +6 ) in the welding fume. 1,2 The U.S. Occupational Safety and Health Administration recently issued a new standard for occupational exposure to hexavalent chromium containing a substantial reduction in the permissible exposure limit, which will make it difficult to weld SS in locations that are not extremely well-ventilated. 3,4 To reduce the emission of Cr +6 from weld fumes, a Cr-free consumable has been developed for welding austenitic stainless steel .5,6 The resulting welds in type 304L SS exhibit mechanical properties and corrosion resistance comparable to welds made with commonly used Cr-bearing consumables.
Because the new consumable has a different chemical composition than the stainless steel base plate to be welded, it is important to consider the effects of galvanic interaction on the localized corrosion behavior of the new consumable. Therefore, two major criteria were established to develop a new weld metal for stainless steel: 5 1. The breakdown and repassivation potentials of the weld metal should be higher than the corrosion potential of the stainless steel base metal in environments of interest to prevent localized attack of the weld metal.
2. The corrosion potential of the weld metal should be slightly higher than that of the stainless steel base metal so that the weld metal is cathodically protected.
Ni-Cu alloys initially were selected based on their galvanic compatibility with types 304 and 316 SS in chloride environments. 5 304L SS was welded with Monel filler metal, which contains 28-34 wt% Cu, to create high-quality welds with no cracks 5 However, segregated regions rich in Cu were weak spots for corrosion susceptibility in the welds .5 Subsequent work on alloys with lower Cu content led to the selection of Ni-10 wt%Cu-1 wt%Pd, which had higher breakdown potential than Monel (though not as high as 304 SS), higher repassivation potential, and slightly higher OCP than 304 SS, and no localized corrosion observed after long term exposure in 0.1 M NaCl. The corrosion properties of the newly developed alloy satisfy the design criteria for a Cr-free filler metal for welding of 304 SS. 6 However, the role of Pd and Cu on the improvement of the corrosion properties has not been clearly understood. Cu has been known to have both beneficial and harmful effects on the corrosion resistance of Ni alloys. The corrosion resistance of Ni-Cu alloys was found to be superior to pure Ni only in the active region, 7 and the optimum content of Cu in Ni-Cu alloys to reduce the corrosion rate was about 30% in 10% HCl, 3% NaCl, and 10% HNO 3 . 8 Verink generated experimental Pourbaix diagrams for Cu -(0-75%)Ni systems in Cl --containing solutions. With decreasing Cu content, the passivity of Ni-Cu alloys was improved in the acidic region. The higher Cu-containing Ni-Cu alloys showed higher weight loss in quiescent seawater after 2 years. 10 A low Cu content in Ni-Cu alloys promoted the formation of a protective Ni(OH) 2 film, and the dissolution of Cu was inhibited in alkaline solution. 11 Although increasing Cu content from 30 through 53 wt% increased the weight loss, it also decreased the pitting depth of Ni-Cu alloys in the same environment .12 After long term immersion, Ni-Cu alloys including Monel 400 showed shallow pits in Cl --containing environments, whereas Cr-containing alloys such as the stainless steels were deeply attacked at local sites .13-17 Isaacs and Newman reported that dissolved Cu was plated out in an artificial pit electrode of type 304 stainless steel in 1 M NaCl solution. 18,19 This reaction occurred around -0.27 V SCE , indicating the reduction of Cu from CuCl2. For Ni-Cu alloys with Cu content from 30-50 wt% in natural seawater, Cu selectively dissolved as Cu + and Cu 2+ , and redeposited as a metallic Cu. 20 The kinetics of the dissolution and reduction of Cu can play an important role in breakdown and repassivation behavior of Ni-Cu alloys.
Pd is not reactive in many environments and is very effective as a hydrogeneration catalyst. 21 The doping effect of the noble Pd on the oxide film of Ni-base alloys was reported to improve the life of the oxide in a deaerated aqueous environment at 288 o C. [22][23][24] Hettiarachchi 25 also found that Pd incorporation into the growing oxide on the surface of austenitic stainless steel improved the resistance to stress corrosion cracking (SCC).
The addition of a noble metal such as Au, Pt, or Pd ennobled the open circuit potential (OCP) of type 316 stainless steel in 1 N H 2 SO 4 solution for 150 h, and enrichment of these noble metals was detected on the surface. 26 The ennoblement of the OCP and low passive/active current densities of Fe-25Cr alloy and Fe-18Cr-10Ni alloy were also reported with the enrichment of Pd on the surface by electrolytic deposition. 27 Potgieter 28 studied the cathodic modification of alloys with noble metals. The noble metals provided a high exchange current density for the reduction reaction, and then shifted the OCP to the passive range resulting in easy passivation. The effectiveness of this modification was best for platinum group metals (PGMs) and was ordered in terms of high effectiveness from Pt to Pd, Ir, Ru, and Os. Potgieter also found that the noble metal was enriched on the surface after active dissolution and that Mo had a synergetic effect with PGMs. From a study of pure Cr, the addition of 0.1% Pd lowered the corrosion rate by 10 5 times, as the PGMs blocked the kink sites where active dissolution preferentially occurred.
In this paper, the influence of Cu and Pd on the passivity and passivity breakdown of Ni is addressed. Passive film composition determined by X-ray photoelectron spectroscopy (XPS), and localized corrosion kinetics determined by artificial pit electrode experiments and proved by a thin foil crevice test, are presented. Figure 1. Schematics of (a) the artificial pit electrode and (b) the electrochemical test procedure.

Experimental
Five Ni alloys were studied: pure Ni, Ni-10Cu, Ni-1Pd, Ni-30Cu, and Ni-10Cu-1Pd (in wt%). The alloys were melted from pure materials and cast in the shape of small disks. By a series of processes, they were worked to wires with 0.17 or 0.34 mm diam. The wires were degreased and annealed at 1100°C for 1 h in vacuum-sealed quartz tubes. Finally, they were mounted in epoxy. The details of the artificial pit electrode are shown schematically shown in Fig. 1. The samples were ground to no. 600 grit and then inserted facing upward into a cell that was filled with 0.1 M NaCl. A Pt wire counter electrode and saturated calomel electrode (SCE) reference electrode were used.
Three electrochemical steps in series were used to evaluate the polarization behavior of the artificial pits. First, to make an artificial pit of a specific depth, a constant potential was applied, 0.8 V SCE for Ni-30Cu and 0.5 V SCE for the rest of the samples. The time to generate a certain pit depth during the potentiostatic test was calculated using Faraday's law on preliminary experimental runs. Four different depths of pits on each sample were tested, where the ratio of the pit depth/diameter was in the range of 0.5 to 4.0. The potential was then stepped down to -0.12 V SCE for 30 s to dissolve the salt film in the pit. 29 Finally, the potential was scanned from 0.3 V SCE to -0.2 V SCE at 10 mV/min. The depths of the artificial pits were confirmed after the experiments by observation in an optical microscope and by integration of the current. Some of the wires were extracted by breaking the epoxy mold after the test and then analyzed using SEM-EDS. Upward scanned potentiodynamic polarization experiments were performed on some samples with no prior dissolution, so that the surface of the electrode was coplanar with the epoxy mount.
Samples of each alloy 10 × 10 mm in size were polarized in 0.1 M NaCl for 10 min at 0 V SCE where all of the samples were in the passive state. After passivation, the samples were rinsed with distilled water and dried. After sitting in the vacuum chamber for about 16 h, the passive films were analyzed using XPS at 13 kV with monochromated X-rays from an Al target. The sample surface was intermittently sputter-etched using Ar ions for 15 or 30 s. The sputtering rate was about 20 Å/min. Two alloys, pure Ni and Ni-10Cu-1Pd, were prepared as foils for crevice corrosion testing. Homogenized 1 mm thick plates were polished down to no. 600 grit abrasive paper until reaching a final thickness of 0.17 mm and electrically connected with a Cu wire. This foil was embedded in epoxy with one edge of the foil exposed at the top surface of the mold. A side of the epoxy mold parallel to the foil electrode was thinned to about 0.17 mm by grinding and polished to 1 µm to facilitate visual observation of the side of the foil (Fig. 2). The foil edge was exposed vertically in a cell containing 0.1 M NaCl and a potential of 0.5 V SCE was applied for 6 h. The configuration of the electrodes and applied potential were the same as those used as in the artificial pit electrode experiments. Furthermore, the foil thickness was the same as the diameter of the wires in the artificial pit electrode. The advantage of the foil crevice electrode was that it allowed observation of the change in topography of the surface during polarization. The side view of the foil sample during propagation of the localized attack downward from the foil edge was recorded using a video camera and VCR.

Results
XPS analysis -XPS analysis was used to evaluate the composition of the prepassivated alloy surfaces. The O 1 s spectra (not shown) indicated that the outermost surface of the passive film for all the tested Ni alloys consisted primarily of hydroxide, and that the inner part of the passive film was mixed oxide and hydroxide. The Pd 3d 5/2 , Cu 2p 3/2 and Cu LMM Auger peaks were analyzed to assess the oxidation states of Pd and Cu. These elements mainly existed in the unoxidized metallic state, but small amounts of PdO and CuO were detected in the outermost region, Fig. 3.
The height of the O 1 s, Cu 2p 3/2 , and Pd 3d 5/2 peaks of Ni-10Cu-1Pd alloy were analyzed as a function of depth during sputter profiling, Fig. 4. It is possible that some Cu +1 is included in the analysis for CuO, but the total amount of oxidized Cu was small. Figure 3 shows that both metallic Cu and Pd were present in the passive film, but in depleted concentrations relative to the content in the bulk alloy. Artificial pit electrode studies - Figure 5 shows the polarization curves for artificial pit electrodes of 0.17 mm diam and similar depths in the range of 0.3-0.4 mm, obtained by scanning downward from 0.3 V SCE as described above. The pit current decreased during the reverse scan as the potential decreased. After reaching a zero-current potential, the current changed polarity becoming negative. The cessation of artificial pit growth can be accomplished by two different processes-repassivation or deactivation. Repassivation occurs when the current density decreases during downward potential scanning and the conditions at the pit bottom change to the point at which the active state can no longer be sustained so the surface repassivates. [30][31][32] This repassivation process is accompanied by a sharp drop in current density to a lower passive current density. The cessation of pit growth during downward scanning can also result from a deactivation process in which the potential is scanned through the corrosion potential of the electrode surface in the pit environment such that the net current becomes cathodic. 30,31 The term -protection potential‖ is generic and encompasses both repassivation and deactivation.
Cessation of the artificial pit electrodes in this study seemed to be a deactivation process occurring as the result of an increase in the cathodic reaction. The addition of 10% Cu decreased the anodic current relative to pure Ni and increased the cathodic current in the   pit, resulting in deactivation and an increase in the protection potential. The addition of more Cu, to 30 wt %, provided no further benefit. However, the addition of even 1 wt % Pd to Ni-10Cu resulted in a large increase in protection potential as a result of a large increase in the cathodic reaction rate inside the pit. Noble metal alloys have previously been found to cause such increases in cathodic reaction rates. 21,33 The cathodic reaction in the artificial pit might come from reduction of dissolved oxygen molecules, protons, or metallic ions. Figure 6 shows pit polarization curves for Ni-10Cu-1Pd in deaerated and aerated solutions. The curves are identical, indicating that the ca-thodic reaction was not oxygen. This was expected, as oxygen should be depleted in the constricted crevice. Furthermore, the protection potential of Ni-10Cu-1Pd alloy was 0.15 V SCE , which is much higher than the reversible potential of H + /H 2 , indicating that hydrogen evolution is probably not the reduction process. However, this potential is very close to the standard equilibrium potential of Cu 2+ /Cu, 0.337 V NHE or about 0.1 V SCE .
In an effort to assess the possibility of cupric ion reduction in the artificial pit electrodes, measurements were made in environments with purposeful additions of cupric ions at a concentration of 0.5 M  using samples with no Cu content, Fig. 7. The protection potential for the alloy Ni-1Pd was 0.1 V SCE in 0.5 M CuCl 2 + 0.1 M NaCl solution, but only -0.4 V SCE in 0.1 M NaCl solution without cupric addition. The protection potential for Ni-1Pd in the cupric-containing solution was close to that for Ni-10Cu-1Pd alloy in 0.1 M NaCl solution with no cupric addition. Pure Ni also showed a very high protection potential in cupric-containing solution, but it was lower than that of Ni-1Pd. The protection potentials for both pure Ni and Ni-1Pd in 0.1 M NaCl with no cupric ion were much lower. These observations suggest that the higher rate of the ca-thodic reaction for Ni-10Cu-1Pd artificial pits in chloride solution was associated with the reduction of the copper ions that were previously dissolved from the alloy into the pit environment. Further evidence for this is given by Fig. 8, which shows the effect of pit depth on the backward-scanned polarization curves for Ni-10Cu-1Pd artificial electrodes of diameter 0.34 mm. For shallow artificial pit electrodes, a sharp decrease in cathodic current of about two orders of magnitude was observed during the back scan. The potential at which this decrease occurred decreased with increasing pit depth. The deepest pit, 1.14 mm deep, was not scanned down far enough to exhibit the current decrease. The sharp decrease is apparently associated with depletion of copper ion from the pit environment. Deeper pits contain a larger reservoir of the dissolved species, and the depletion occurs at a lower potential, after more cathodic charge has passed and more deposition has occurred.
After the first potentiostatic step at 0.5 V SCE in 0.1 M NaCl solution, the epoxy mold around a Ni-10Cu-1Pd sample was mechanically broken and the alloy wire was extracted. The top surface of the wire, which was actually the bottom of the artificial pit electrode, was coated with carbon and analyzed using scanning electron microscopy-energy-dispersive spectroscopy (SEM-EDS), Fig. 9. For Ni-10Cu-1Pd with a pit depth of 0.34 mm, the surface was flat with some scattered crystallographic pits. Local enrichment of Pd and Cu was detected at the same sites across the pit surface. A small amount Cl was also detected at those sites. It has been reported that Cu and Cl can form compounds in pits. 18,19 To monitor the localized attack morphology, a dissolving thin foil crevice sample was viewed from the side face as shown in Fig. 2. A constant potential was applied to pure Ni and Ni-10Cu-1Pd alloy for 6 h in 0.1 M NaCl solution while recording the morphology of the foil from the side. For pure Ni at 0.5 V SCE , breakdown occurred at the two edges of the foil after 1 h and then propagated into the depth. The middle of the foil was unattacked and the surface remained at the position of the original solution/metal (S/M) interface, Fig. 10. For Ni-10Cu-1Pd alloy tested at 0.5 V SCE , the whole surface of the foil was attacked after 1 h and then the S/M interface gradually shifted down with time, Fig. 11. The S/M interface still remained almost as flat as the attack proceeded by repeated breakdown and fast deactivation of many sites. The Pd addition changed the mode of pit propagation from localized growth of few sites to wide spreading of many sites.

Discussion
Previous work showed that the alloy Ni-10Cu-1Pd exhibited superior resistance to localized corrosion in potentiodynamic testing and exposure tests, and is a suitable alloy for use as a Cr-free consumable in the welding of 304 SS. 6 The reasons for this corrosion resistance were explored in this work.
The beneficial effects of noble Pd additions on the passivity of other Ni-alloys has been reported previously. 22,23 However, the results of this study suggest that the addition of Cu and Pd to Ni has little effect on the passive film and that improvement in localized behavior obtained by alloying is more related to the effects on the anodic and cathodic kinetics in pits than to improvements in the protectiveness of the passive film.
It was shown that Ni artificial pit electrodes scanned downward from high potentials exhibit high zero-current potentials when Cu and Pd are alloyed at low levels or when cupric ions are introduced into the electrolyte. The cessation of artificial pit growth is considered to be a deactivation process resulting from a dramatic increase in the cathodic kinetics in the pit. The cathodic reaction was shown not to be oxygen reduction or hydrogen evolution and it can be concluded that the primary cathodic reaction in the artificial pit electrodes was reduction of Cu ions. The great enhancement in the rate of this reaction for the Ni-10Cu-1Pd alloy relative to the Ni-Cu binary alloys and the co-location of Pd and Cu on the surface of an artificial pit electrode suggest that the Cu reduction was catalyzed at sites where Pd was enriched at the surface, perhaps by surface enrichment from the dissolution of Ni and Cu. The synergism between cupric ions in solution and Pd enrichment on the surface resulted in a strong ennoblement of the deactivation process, which decreased the stability of the pits.
Pd also affects the anodic pit kinetics. Comparison of the artificial pit polarization curves for pure Ni and Ni-1Pd in the cupric free 0.1 M NaCl solution (Fig. 7) indicates that in the absence of Cu, Pd results in a large increase in the Ni active dissolution kinetics. In this cupric-free solution, the primary cathodic reaction is probably hydrogen evolution, and there is no obvious effect of the Pd addition on the rate of this reaction. However, the rate of anodic dissolution at a given potential is increased by many orders of magnitude for the Ni-1Pd alloy. The curves in Fig. 5 indicate that this enhancement of Ni dissolution by Pd was not observed for the Ni-10Cu alloy. However, Fig. 8 shows that the anodic portion of the back-scanned polarization curves for Ni-10Cu-1Pd was dependent on the depth of the pit. The deeper pits with depth of 0.6 and 1.14 mm exhibited much higher dissolution rates at a given potential than the shallow pits with depth of 0.12 and 0.27 mm. It is likely that the extent of Pd enrichment increased for the deeper pits, so the effect of Pd on the anodic kinetics in the pits was more evident for the deeper pits. Pd accelerates the rate of anodic dissolution, but this is more than compensated by the strong effect of Pd on Cu deposition, which results in a very high pit corrosion potential. The mechanism for Pd enhancement of Ni dissolution is not understood.
The creation of a deep artificial pit by forced dissolution at high potential results in the formation of a pit environment that could be different than for a pit that grows at a lower opencircuit potential, so the relevance of the artificial pit electrode measurements to pitting at open circuit must be considered. The breakdown and repas-sivation potentials for Ni-10Cu-1Pd in 0.1 M NaCl were found to be + 220 and +150 mV SCE, respectively. A study of the behavior of Monel-400 (with ~30% Cu) in deaerated chloride solution found that both Cu(I) and Cu(II) were generated during anodic dissolution in a wide range of potential, from -350 to + 400 mV Ag/AgCl. 34 At potentials above +150 mV Ag/AgCl (+130 mV SCE), dissolution was dominated by the formation of Cu(II). Therefore, the environment created during the formation of the recess in the artificial pit electrode should be similar to that formed in pits at the breakdown potential measured in cyclic polarization experiments. Figure 12 shows polarization curves measured from the cross sections of 0.17 mm wires that were not pre-dissolved to form deep artificial pit electrodes, i.e., the surfaces of the wires were coplanar with the epoxy mount. The potential was scanned at 10 mV/min in 0.1 M NaCl solution, with a vertex current density of 0.5 mA/cm 2 . The average depth of attack during the cyclic scan was much less than the depth of the artificial pit electrodes and the potentials were much lower. The breakdown potentials for pure Ni, Ni-10Cu, and Ni-10Cu-1Pd were all relatively close. Note that the breakdown potentials of the small wire electrodes were lower than for samples with large areas. However, the backscanned portions of the curves for the flat wire cross sections were vastly different from each other. The pure Ni sample exhibited a large hysteresis and a zero-current potential on the backscan equal to the value on the forward scan. The Ni-10Cu-1Pd exhibited a very small hysteresis and much higher zero-current potential on the backscan. The behavior of Ni-10Cu was intermediate. Clearly, the Cu and Pd contents exerted a strong influence on these curves, even with relatively small extents of attack.
The general understanding of critical potentials is that localized corrosion can initiate at potentials above the breakdown potential, and it can continue to grow at potentials above the protection potential. 35 In real applications under open-circuit conditions, localized corrosion initiates when the potential is high, and then the potential drops as a result of the activation at the pit bottom. Therefore, the environment generated in a pit growing at high open-circuit potentials is relevant to the possible cessation of pit growth at lower open circuit potentials.
Newman and Isaacs performed cyclic voltammetry measurements on small Pt wires inserted into an artificial pit electrode made from a commercial 304L SS wire of larger diameter. 19 They observed oxidation and reduction peaks at -200 and -350 mV SCE, respectively, which they assigned to the Cu/CuCl 2 redox reaction associated with the 0.33% Cu in the alloy. As mentioned above, the protection potential for the Ni-10Cu-1Pd artificial pit electrode is much higher than these values and close to the standard potential for Cu/Cu 2+ . The lower protection potential found for the flat Ni-10Cu-1Pd wire electrode is still higher than the peaks observed by Newman and Isaacs and might be connected to lower cupric ion concentrations. Our interpretation is that the cathodic reaction for the Cu-containing alloys is the reduction of Cu 2+ and that it is relevant to the behavior of these alloys at open circuit conditions.
There have been many studies reporting the superior resistance of Cu-containing Ni alloys to localized corrosion in chloride environments for more than 30 years. These alloys usually showed much shallower pits than stainless steel even if the first formation of the pits was earlier for Ni-Cu alloys than for stainless steels. [13][14][15][16] Ni-10Cu-1Pd alloy in this study also showed very shallow pits in the long term crevice corrosion test, compared to type 304L stainless. 36 This behavior can be explained by the stifling of pit growth by the plating of Cu from the pit environment.

Conclusions
The effects of Cu and Pd on the passivation and localized corrosion behavior of Ni alloys were investigated using XPS and the artificial pit electrode technique. The following are the important conclusions of this study.
The passive film on Ni-10Cu-1Pd alloy consisted of a mixture of oxide and hydroxide, with the outermost layer being mainly hydroxide. Very little Cu and Pd were found in the passive film and they were mostly in the metallic state. This small change in the composition of passive film cannot explain the improved resistance to the localized corrosion of Ni-Cu-Pd alloy.
The addition of 1 wt % Pd increased the artificial pit electrode protection potential considerably over the whole range of the pit depth studied. This protection potential for the Ni-10Cu-1Pd artificial pit electrode is close to the standard potential for Cu/Cu 2+ .
The improved resistance to the localized corrosion of Ni-10Cu-1Pd alloy is attributed primarily to the cessation of pit growth by enhanced reduction of copper ions from the pit environment in the presence of Pd.


extfieldsofstudy: Chemistry Medicine

text:
Folding and Unfolding in the Blue Copper Protein Rusticyanin: Role of the Oxidation State

The unfolding process of the blue copper protein rusticyanin has been studied from the structural and the thermodynamic points of view at two pH values (pH 2.5 and 7.0). When Rc unfolds, copper ion remains bound to the polypeptide chain. Nuclear magnetic resonance data suggest that three of the copper ligands in the folded state are bound to the metal ion in the unfolded form, while the other native ligand is detached. These structural changes are reflected in the redox potentials of the protein in both folded and unfolded forms. The affinities of the copper ion in both redox states have been also determined at the two specified pH values. The results indicate that the presence of two histidine ligands in the folded protein can compensate the change in the net charge that the copper ion receives from their ligands, while, in the unfolded protein, charges of aminoacids are completely transferred to the copper ion, altering decisively the relative stability of its two-redox states.

INTRODUCTION
Copper is essential for life. It participates in many biological events [1][2][3]. After iron, nature has chosen this metal ion as the second element in participating in electron transfer chains [4][5][6]. Its thermodynamics and the kinetic easiness to oxidize/reduce itself allow copper containing proteins to play a crucial role as electron carriers in many vital processes. Copper proteins possess well-designed active centers that finely tune metal ion redox properties [7,8]. In turn, ligands, geometries, and surroundings of the copper ion are a direct consequence of protein scaffold. Therefore, protein folding stabilizes one copper oxidation state versus the other one, resulting in a metal ion biologically active. Protein redox potentials are a consequence of the different affinities of the polypeptide chain for the metal ion in both redox states [9,10]. It follows the relevance of studying the formation of the functional (folded) holoprotein, in both the reduced and the oxidized states, from the structural and functional points of view. Taking into account that the holoprotein can be formed as an unfolded protein prior to copper uptake inside the cell [11], it also follows the relevance of the same kind of studies with the unfolded protein. This last point is still a subject of debate [10,12].
Blue copper proteins (BCPs) are relatively small soluble copper proteins [7,14,15]. Their characteristic colour arises from a charge transfer ligand-to-metal band between a cysteinyl sulphur atom and the copper(II) ion [8,16]. All BCPs present the so-called Greek key β-barrel as a common feature (Figure 1(a)). This is a rigid structure essentially kept by an extended network of hydrogen bonds and tertiary interactions between amino acid side chains. This rigidity is transmitted to the metal ion and is essential for the electron transfer. The copper ion is bound to a cysteine and two histidine residues. A fourth ligand completes the copper coordination sphere for most BCPs [17]. This fourth ligand is usually a methionine (Figure 1(b)). In all BCPs, the ligands Cys, Met, and one of the histidines (hereafter called HisC, due to its proximity to the C-terminal end) are close among them in the primary sequence, while the other histidine, HisN, is far away from them in the amino acid chain (Figure 1(b)). Table 1 shows the redox potentials of some representative BCPs [13]. They are all encompassed between 120 and 620 mV, while that of the pair Cu(II)/Cu(I) in aqueous solution is 150 mV, substantially lower than most of them. Thus,  these proteins considerably increase the stability of the reduced versus the oxidized state of the copper ion. Rusticyanin (Rc) is, by far, the BCP with the highest capability in this respect. Rc is the most abundant soluble protein of the Gramnegative bacterium Acidithiobacillus ferrooxidans (Af ) [18][19][20]. This organism lives in media that are very acidic and containing high concentrations of metals. Consequently, Rc is extraordinarily stable at low pH values. According to the well-established "rack state" [1] or "entatic" [21] mechanism, protein folding in BCPs imposes a coordination geometry atypical to the copper(II) ion. Oxidized copper would then be destabilized, increasing the redox potential of the metal ion. Nevertheless, the BCP azurin presents a redox potential in the unfolded state higher than that of the folded protein (450 versus 276 mV) [10,22]. So, apparently, the unfolded protein stabilizes the reduced copper in a more efficient way, and protein folding seems not to be so crucial in keeping the redox potential of this BCP.
Here we present an exhaustive study of the unfolding process of rusticyanin by means of guanidinium chloride (GdmCl). We have previously characterized the structural facets of the Rc unfolding process [12,23,24]. We expose now the parallel changes produced in the Rc redox potential (i.e., the different affinity of the metal ion in the two redox states). Rc retains copper(I) and copper(II) in the unfolded form. Moreover, the redox potential of this pair for the unfolded Rc is slightly higher at low pH values than the analogous pair for the folded form. We demonstrate that the folding scaffold not only rises the protein affinity for the metal ion in whatever oxidation state, but also, and mainly, hampers drastic changes in its redox properties when pH is modified.

Protein preparation
Recombinant rusticyanin was obtained from BL21(DE3) Escherichia coli containing the Rc plasmid [19]. Bacteria cultures were grown in suitably modified M9 medium [25]. Samples for all techniques were prepared as previously described [25]. Conditions for all experiments (unless otherwise indicated) were formiate (pH 2.5) or phosphate (pH 7.0) buffers 10 mM, 298 K.

Uv-visible and nuclear magnetic resonance spectroscopies
Uv-visible data were collected in a UV-visible spectrophotometer (Shimadzu UV-1603). Copper titrations were carried out starting from the apoprotein (2-5 ×10 −5 M) and adding the corresponding aliquots of copper solutions up to protein saturation. Buffers used were formic acid 10 mM and Tris chloride 10 mM at pH 2.5 and 7.0, respectively. For the folded protein, copper(II) titrations were performed by following the absorption band at 592 nm (ε = 2240 M −1 cm −1 ) [26]. For the unfolded protein, the Cu(II)Rc Cu(I)Rc Scheme 1: Thermodynamic cycle associated to the redox potential of CuRc. Redox potentials (equations 1 and 2) are given versus the normal pair H + , H 2 in standard conditions. Copper(II) redox potential (equation 2) is 150 mV (ΔG 0 = −3.45 kcal/mol). All values were determined at 298 K.
formation of the complex was followed by the absorbance at 270 nm (ε = 2000 M −1 cm −1 ) due to the copper(II)-SγCysband charge transfer band, as we have previously described [12]. This absorbance is added to the intrinsic absorption of rusticyanin at 280 nm. The affinity constants of rusticyanin for the reduced species were deduced applying the thermodynamic cycle shown in Scheme 1 (see Section 3). Throughout all the text, values of equilibria 3 and 4 are always referred to the formation of the corresponding holoforms (affinity constants) and, consequently, they are given in units of M −1 . All experiments were performed at 298 K.
In nuclear magnetic resonance (NMR) measurements, eight percent of D 2 O was added to the samples used (phosphate buffer 10 mM, pH 5.5, 298 K). All the experiments were performed as described in [25].

Cyclic voltammetry
Voltammetric experiments were carried out with an Autolab PGSTAT30 (Echo Chemie) in a low volume (∼3 mL) electrochemical cell with a three-electrode configuration [27]. The working electrode was either a gold disk modified with 4-mercaptopyridine (measurements at pH 7.0), or a carbon paste surface modified with 4,4 -bipyridyl (measurements at pH 2.5). Electrolyte solutions contained 0.1 M sodium phosphate buffer, 0.1 mM rustcyanin, and, in the studies of the unfolded protein, 8 M guanidinium chloride. All measurements were carried out at 25 ± 0.2 • C. Given electrode potential values are referred to the normal hydrogen electrode (NHE) scale. Signals were reversible both for folded and unfolded forms. The estimated error of potential measurement was lower than ±15 mV in all cases (the errors of each measurement are specifically given in Section 3).

The unfolding process: structural and dynamic characterization
We have previously characterized the chemical unfolding process of Rc by different biophysical techniques [12,23,24].
NMR permits to obtain dynamical and structural information not provided by other spectroscopies. In fact, diffraction crystallography cannot be applied to partially or totally protein unfolded states and then NMR becomes the most powerful structural technique. Figure 2 shows the 1 H− 15 N HSQC spectra obtained from three solutions containing apoRc, Cu(I)Rc, and Cu(II)Rc after adding GdmCl until complete protein unfolding. The three spectra are nearly superimposable, indicating that, essentially, the same species are present in the three samples. When comparing apoRc and Cu(I)Rc (Figure 2(a)), a set of few signals (labelled as a − i) modify their chemical shifts. These small local changes indicate that copper(I) remains bound to the unfolded rusticyanin. In the 1 H− 15 N HSQC spectrum of copper(II) rusticyanin (black line, Figure 2(b)), the intensities of the same signals (a−i) are markedly diminished, or they are even lost, due to the paramagnetism of copper(II) [28]. Thus, the metal ion is bound to the unfolded polypeptide chain at the same residues in whatever oxidation state. Unfortunately, at this stage, the low resolution of the spectra did not allow us to unequivocally identify the specific residues bound to the copper ion. The redox activity of the unfolded rusticyanin (see below) also demonstrates that copper is bound to this form of the protein. Figure 3 shows the 1 H− 15 N HSQC of Cu(I)Rc at different guanidinium chloride concentrations. In absence of the denaturant agent, only the folded species (characterized by a large dispersion of the signals) is present (Figure 3(a)). On the contrary, only the unfolded species are observed at GdmCl 6 M (Figure 3(c)). At GdmCl 2.1 M (Figure 3(b)), both species are present in different ratios (ca. 3 : 2 for the folded versus the unfolded species, resp.). At this step, the folded species essentially has the same chemical shifts as the completely folded species, indicating that the tridimensional protein structure is kept. We have determined the mobility features of this species. Figure 4 displays the comparison of the relaxation data (R 1 , R 2 , and 1 H− 15 N NOE) for the completely folded Rc and the species present at a GdmCl 2.1 M concentration. As observed, folded Rc at these conditions is much more mobile than the completely folded species (i.e., in absence of GdmCl). In other words, GdmCl induces the opening of Rc tridimensional structure.
Two main conclusions are immediately deduced from the relaxation data. First, with the exception of the N-terminal domain, the largest mobile region of Cu(I)Rc at GdmCl 2.1 M is that encompassing residues 80-120 (Figure 4(b)). These residues correspond to a long loop present not only in Rc, but also in all BCPs. It is interesting to remark that the fist copper ligand in the primary sequence, HisN (His85 in Rc, Figure 1(a)), belongs to this loop [29]. The dynamic properties of this ligand itself indicate that this histidine undergoes exchange processes. Deuterium exchange experiments also indicate that amide protons of this domain are highly exposed to the solvent in these conditions (data not shown), corroborating that this loop is prone to open. By resuming, HisN is more labile when the protein starts to unfold.
On the contrary, the C-terminal domain (amino acids 120-155, encompassing the last two β-strands) is one of the most rigid domains of the protein even when GdmCl is added (Figure 4(b)). Moreover, most of the amide protons in these regions essentially remain unchanged upon addition of GdmCl (data not shown). Both data indicate that the extreme C-terminal is one of the most protected domains of Rc, even in the presence of the denaturant agent. The three last ligands of the metal ion (Cys138, His143, and Met148, see Figure 1(b)) belong to this domain [29]. In summary, when the denaturant agent is added, the interaction of the copper ion with HisN starts to weaken, while that kept with the other three ligands is not essentially altered. This strongly suggests that these three ligands remain bound to the metal ion when the protein unfolds, while HisN is lost from the copper coordination.
This conclusion is totally supported by comparison between the present results and previous data obtained from the BCP azurin. In fact, the unfolding process of azurin has been amply studied [10,30,31]. It has been demonstrated that HisN in azurin is detached from the copper ion when protein unfolds, while the other three ligands remain bound to it. Moreover, the redox potential of the unfolded rusticyanin (450 mV, see below) and azurin (456 mV, [22]) is the same within the experimental error, indicating that copper possesses a similar coordination sphere in both unfolded proteins. We can conclude that the same process occurs for these two similar BCPs. When Rc (or azurin) unfolds, copper ion remains bound to the three ligands close in the primary sequence (Cys, HisC, and Met), two of which belong to defined secondary structure elements of the protein (the last two β-strands of the β-barrel, see Figure 4). On the contrary, the first ligand (HisN), far away in the primary sequence and located in a flexible loop of the protein, detaches from copper upon unfolding. Figure 5 displays a schematic view of copper coordination in unfolded Rc.

Functional aspects: redox potentials, and relative copper-protein affinities
Values of redox potentials of a holoprotein reflect the different affinity of the apoprotein for the two redox states of a metal ion. The corresponding thermodynamic cycle applied to CuRc is shown in Scheme 1. We have studied the equilibria of this cycle in both folded and unfolded forms of Rc and at two different pH values (2.5 and 7.0). Redox potentials were measured by means of cyclic voltametry. The cyclic voltammograms are displayed in Figure 6. At pH 2.5, the redox potential of the pair Cu(II)Rc/Cu(I)Rc (equilibrium 1) was 620 ± 10 mV, the same within the error previously reported [32]. The affinity constant of Cu(II) for apoRc (equilibrium 3) was measured by visible spectroscopy, following the formation of the blue charge transfer band characteristic of Rc at 592 nm (Figure 7 Cycle of Scheme 1 can also be applied to the unfolded Rc at the same pH value (2.5). The redox potential of the pair Cu(II)/Cu(I)Rc for the unfolded protein has been measured, resulting to be 630 ± 10 mV (ΔG 0 3 = −14.7 kcal/mol). This value is slightly higher than that of the folded protein, indicating that, in these conditions, protein scaffold is less efficient in stabilizing copper(I) than the unfolded protein. The affinity constant of copper(II) for the unfolded form of Rc was determined by following the charge transfer band that appears at ca. 270 nm [12]. The obtained value was 500 ± 60 M −1 for the corresponding affinity constant (Figure 7(b)). Then the affinity constant of unfolded Rc for the reduced (copper(I)) species (equilibrium 4) can be deduced by closing the cycle. The resultant value was 6.55 × 10 10 M −1 . Figure 8 shows the relative stabilities of the different holo species.
An analogous study was carried out at pH 7.0. The redox potential of the folded and unfolded forms of Rc are 550 (±5) and 450 (±15) mV, respectively. These values substantially decrease with regard to those obtained at pH 2.5 (see above). The redox potentials of folded Rc with the pH have been previously reported [32]. They are coincident with those here obtained. On the other hand, it is interesting to remark that redox potential of the unfolded rusticyanin at neutral pH is essentially the same to that obtained for unfolded azurin at the same experimental conditions (456 mV, [22]). This also corroborates that both azurin and rusticyanin possess the same coordination sphere in their unfolded forms.
Applying the same cycle (Scheme 1), the corresponding affinity constants can be obtained. In Figure 8, these values are displayed as well. As observed, the decrease in the redox potential at neutral pH is an immediate consequence of the high increment in the affinity of Rc for the copper(II) ion, from −4.8(±0.4) to −11.4(±1.0) kcal/mol and from −3.7(±0.5) to −9.4(+1.0) kcal/mol, for the folded and unfolded forms, respectively. This decrease is the result of a protonation/deprotonation residue close or, overall in the case of the unfolded protein, bound to the copper ion. From  the copper ligands, only a histidine residue can undergo such a process in this pH range. Then the deprotonation of a histidine (necessarily HisC in the unfolded protein) is responsible for the decrease of the redox potential when the pH increases. In fact, a deprotonated histidine (with a lower positive charge) would present a higher affinity for the copper(II) ion, decreasing the redox potential. It is interesting to remark that the folded species shows a manifestly higher affinity for the copper ion (in whatever oxidation state) than the unfolded form. This is more evident at pH 7.0 (see Figure 8). Rusticyanin is synthesized at the cytoplasm from Af at pH 6.5 and then secreted to the periplasm (pH ca. 3.0) [33]. Then, the present result can be relevant in vivo since free copper is found in an extremely low concentration inside the cell [2].
Finally, it is also noteworthy that the decrease in the redox potential is much more acute for the unfolded than for the folded form. This is a result of the different variation in the affinity of the reduced copper for the folded and unfolded Rc species (see Figure 8). While Cu(I) substantially increases its affinity for folded Rc when the pH rises, its affinity for unfolded Rc is not substantially altered. As a result, a drastic variation of the redox potential with the pH is observed. Then protein scaffold is acting as a "buffer" against a change in the charges of the media (i.e., aminoacids coordinated to the metal ion). When undergo protonation/deprotonation processes, the two histidines bound to the copper ion can interact with nearby residues of the active site, decreasing their net charge. Consequently, modifications in copper charge due to pH variations are also diminished.  Figure 8: Gibbs free energies (in kcal/mol) corresponding to the formation of the complexes between copper (in both redox states) and the apoprotein (in the folded and unfolded forms), that is, corresponding to the equilibria (3) and (4) of Scheme 1. Light and dark grey bars correspond to data at pH 2.5 and pH 7.0, respectively. For the sake of clarity, the values of ΔG 0 associated to each process are explicitly indicated at the bottom of each bar.