Active-Site Protonations

With [Co(phen)3] + as oxidant for PCu(I), rate constants determined by the stopped-flow method approach zero at low pH, consistent with zero reactivity of the trigonally coordinated Cu(I) form. However, with [FelCNlel as oxidant there is sometimes difficulty in fitting rate constants to the relevant [H ] dependence, which leaves open the question as to whether the rates actually become zero (57). Whereas [Co(phen)3l + is believed to react with PCu(I) at both the remote (acidic) and the adjacent (hydrophobic) patches, [FefCNleP reacts predominantly at the latter. The instability of PCu(I) in solution at pH 4.5 makes it difficult to settle this issue conclusively. The range of studies has been extended using the pH-jump method in which protein, at high pH (with relatively small concentration of buffer), is stopped-flow mixed with the redox reagent at low pH (with excess buffer), and this approach has been used more extensively in recent studies. 

Of the other blue copper proteins, only amicyanin shows a similar effect of pH (79), and a TpK of 7.18 has been obtained for the Cu(I) state. As with plastocyanin, no corresponding effect is observed for Cu(II) amicyanin, at least down to pH 4.5. The physiological relevance in the case of both proteins is at present unclear. Because in photosynthesis the pH of the inner thylakoid is less than 5.0, one possibility is that this is related to proton transport. Alternatively, it quite simply may be a control mechanism for electron transport. 

Crystal structure information (4) for poplar plastocyanin in the Cud) state at low pH has indicated the existence of two conformers. The 

Fi(i 10. The H -induced dissociation of Cu(I)—NiHis 87) of plastocyanin (and amicyanin), and existence of two conformers of the protonated form (4). 

The H -induced changes at the amicyanin active site have been studied kinetically by NMR (68). The reaction scheme (in D2O) can be written as in Eq. (2), 

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Table 2. Acid dissociation pK values, 1 = 0.10 M(NaCl), relating to the active site protonation of different plastocyanins, PCu(I), as determined by (a) proton NMR (b) the variation of rate constants (25 °C) with pH for the [FelCN) ] oxidation of PCu(I), 1 = 0.10 M(NaCl), and (c) similar experiments with [Co(phen)3] " as oxidant. The latter is an apparent value only, and is believed to be composite due to reaction occurring at the remote site...

It is appropriate now to return to the effect of pH on the [Co(phen)3] oxidation of PCu(I). If protonation at the remote site influences the reaction of PCu(II), then a similar effect might be expected for the reaction of PCu(I) with positively charged complexes. In the case of PCu(I) the kinetics are dominated by the inactivation resulting from the active site protonation. Whereas the pK for the [Fe(CN)g] oxidation is in good agreement with the HNMR independently measured value, the apparent pK obtained with [Co(phen)3] " is significantly higher, an effect which is clear from an inspection of Fig. 10. A two pK fit is possible in the case of [Co(phen)3], as has been illustrated [1,100],... 

The important processes occurring in a catalyst layer include interfacial ORR at the electrochemically active sites, proton transport in the electrolyte phase, electron conduction in the electronic phase (i.e., Pt/C), and oxygen diffusion through the gas phase, liquid water, and electrolyte phase. 

Active site protonations, blue copper proteins, 36 396-398... 

Blue copper proteins, 36 323, 377-378, see also Azurin Plastocyanin active site protonations, 36 396-398 charge, 36 398-401 classification, 36 378-379 comparison with rubredoxin, 36 404 coordinated amino acid spacing, 36 399 cucumber basic protein, 36 390 electron transfer routes, 36 403-404 electron transport, 36 378 EXAFS studies, 36 390-391 functional role, 36 382-383 occurrence, 36 379-382 properties, 36 380 pseudoazurin, 36 389-390 reduction potentials, 36 393-396 self-exchange rate constants, 36 401-403 UV-VIS spectra, 36 391-393 Blue species... 

UV-VIS Spectra Reduction Potentials Active-Site Protonations Charge on Proteins Self-Exchange Rate Constants Electron Transfer Routes Comparison with Rubredoxin Summary References... 

Although the Cu(II) form of T. versutus amicyanin is stable down to pH 4, the reduced form begins to denature below pH 6.4 (63). An active site protonation of the Cu(I) protein similar to that of plastocyanin, but with pKa 7.18, affects the reduction potential. 

Henderson et al. [223] presented a detailed pattern of the structure of bacteriorhodopsin using high-resolution cryoelectron microscopy. Using X-ray and neutron diffraction techniques, Dencher et al. [224—227] could decode the secondary and tertiary structure of bacteriorhodopsin during the photocycle. Nevertheless, we should emphasize that the resolution still shows transitions in the active site (protonation of counterions, deprotonation of Schiff base, and reprotonation of counterions), leading to a metastable state of the protein. 

Cummins, P. L., Gready, J. E. (2001) Energetically most likely substrate and active-site protonation sites and pathways in the catalytic mechanism of dihydrofolate reductase, J. Am. Chem. Soc. 123, 3418-3428. 

Distances between the nitroxide of bound steroid and several assigned protons in or near the active site could be determined on the basis of the paramagnetic effects of the nitroxide on the relaxation rates of the active site protons. These distances could then be used to dock the structure of the spin-labeled steroid into a partially refined 2.5-A resolution X-ray structure of the isomerase 128). However, in order to rationalize the chemical evidence that Asp-38 is involved in the catalytic mechanism, it was necessary to assume that steroid substrates and the spin-labeled steroid (16) are bound not only with reversal of the C-3 and C-17 positions, but also with reversal of the planes of the steroid ring systems ( up-sidedown binding), positioning Asp-38 above the C-4j8 and C-6j3 protons. 

X. Chen and A. Tropsha, /, Med, Chem., 38, 42 (1995). Relative Binding Free Energies of Peptide Inhibitors of HIV-1 Protease The Influence of the Active Site Protonation State. 

Ft (platinum) catalysts supported on a conductive matrix, such as carbon, to provide electron conduction and (3) a hydrophilic agent, such as polytet-rafluoroethylene (PTFE) to provide sufficient porosity and adjust the hydro-phobicity/hydrophilicity of the CL for gaseous reactants to be transferred to active sites [2,3]. With each of those elements optimized to provide the best overall performance, the CL functions as a place for electrochemical reactions. The processes occurring in a CL include mass transport of the gaseous reactants, interfacial reactions of the reactants (e.g., H2 at anode and O2 at cathode) at the electrochemically active sites, proton transport in the electrolyte phase, and electron conduction in the electronic phase. When contaminants are present in the reactant streams, one or more of the above processes can be adversely affected, causing degradation in fuel cell performance or even fuel cell failure. 

This section details the precise steps necessary for the calculation of the relative binding free energy, A(AG ), and its components as given by equation (24). First the active site protonation state is determined. It is difficult to experimentally determine a specific protonation state for each aspartic acid (ASP 25, ASP 125) [49] present in the isolated HlV-1 protease binding pocket. That is because in the isolated enzyme these two aspartic acids are chemically equivalent and proximate so the protonation state of one is inexplicably correlated with that of the other. Titration studies yield two pK values [49, 50] however these are associated with the ASP 25, 125 pair as a whole. 

Given the active site protonation states as determined above, the structures of LP, LP j, P, P that go into the free energy calculations defined by equation (23) or (24) were determined using the following protoeol (this optimization protocol is also displayed in Figure 4) ... 

F. Sussman, M. C. Villaverde, J. L. Dominguez and U. H. Danielson, On the Active Site Protonation State in Aspartic Proteases Implications for Drug Design, Curr. Pharm. Des., 2013,19, 4257. 

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