Examination of n=2 reaction mechanisms that reproduce pH-dependent reduction potentials.

Flavo- and quinoproteins often exhibit trends in reduction potentials that approximate -30 mV per pH unit. At least five different reaction mechanisms can model the behavior described by the following Nernst equation: E(observed)=E0'+(0.059/2) log [H+]. The pH-reduction potential profile described by this equation can be reproduced by various models using different reaction mechanisms, and these mechanisms yield different reduction potentials and acid dissociation constants. In order to understand these discrepancies, this article discusses how the various methods reproduce this pH-dependent Nernst equation.

Cytochrome b5 reductase: role of the si-face residues, proline 92 and tyrosine 93, in structure and catalysis.

The conserved sequence motif "RxY(T)(S)xx(S)(N)" coordinates flavin binding in NADH:cytochrome b(5) reductase (cb(5)r) and other members of the flavin transhydrogenase superfamily of oxidoreductases. To investigate the roles of Y93, the third and only aromatic residue of the "RxY(T)(S)xx(S)(N)" motif, that stacks against the si-face of the flavin isoalloxazine ring, and P92, the second residue in the motif that is also in close proximity to the FAD moiety, a series of rat cb(5)r variants were produced with substitutions at either P92 or Y93, respectively. The proline mutants P92A, G, and S together with the tyrosine mutants Y93A, D, F, H, S, and W were recombinantly expressed in E. coli and purified to homogeneity. Each mutant protein was found to bind FAD in a 1:1 cofactor:protein stoichiometry while UV CD spectra suggested similar secondary structure organization among all nine variants. The tyrosine variants Y93A, D, F, H, and S exhibited varying degrees of blue-shift in the flavin visible absorption maxima while visible CD spectra of the Y93A, D, H, S, and W mutants exhibited similar blue-shifted maxima together with changes in absorption intensity. Intrinsic flavin fluorescence was quenched in the wild type, P92S and A, and Y93H and W mutants while Y93A, D, F, and S mutants exhibited increased fluorescence when compared to free FAD. The tyrosine variants Y93A, D, F, and S also exhibited greater thermolability of FAD binding. The specificity constant (k(cat)/K(m)(NADH)) for NADH:FR activity decreased in the order wild type > P92S > P92A > P92G > Y93F > Y93S > Y93A > Y93D > Y93H > Y93W with the Y93W variant retaining only 0.5% of wild-type efficiency. Both K(s)(H4NAD) and K(s)(NAD+) values suggested that Y93A, F, and W mutants had compromised NADH and NAD(+) binding. Thermodynamic measurements of the midpoint potential (E degrees ', n = 2) of the FAD/FADH(2) redox couple revealed that the potentials of the Y93A and S variants were approximately 30 mV more positive than that of wild-type cb(5)r (E degrees ' = -268 mV) while that of Y93H was approximately 30 mV more negative. These results indicate that neither P92 nor Y93 are critical for flavin incorporation in cb(5)r and that an aromatic side chain is not essential at position 93, but they demonstrate that Y93 forms contacts with the FAD that effectively modulate the spectroscopic, catalytic, and thermodynamic properties of the bound cofactor.

Crystallographic studies of V44 mutants of Clostridium pasteurianum rubredoxin: effects of side-chain size on reduction potential.

Understanding the structural origins of differences in reduction potentials is crucial to understanding how various electron transfer proteins modulate their reduction potentials and how they evolve for diverse functional roles. Here, the high-resolution structures of several Clostridium pasteurianum rubredoxin (Cp Rd) variants with changes in the vicinity of the redox site are reported in order to increase this understanding. Our crystal structures of [V44L] (at 1.8 A resolution), [V44A] (1.6 A), [V44G] (2.0 A) and [V44A, G45P] (1.5 A) Rd (all in their oxidized states) show that there is a gradual decrease in the distance between Fe and the amide nitrogen of residue 44 upon reduction in the size of the side chain of residue 44; the decrease occurs from leucine to valine, alanine or glycine and is accompanied by a gradual increase in their reduction potentials. Mutation of Cp Rd at position 44 also changes the hydrogen-bond distance between the amide nitrogen of residue 44 and the sulfur of cysteine 42 in a size-dependent manner. Our results suggest that residue 44 is an important determinant of Rd reduction potential in a manner dictated by side-chain size. Along with the electric dipole moment of the 43-44 peptide bond and the 44-42 NH--S type hydrogen bond, a modulation mechanism for solvent accessibility through residue 41 might regulate the redox reaction of the Rds.

Voltammetric simulations of multiple electron transfer/proton transfer coupled reactions: flavin adenine dinucleotide as a model system.

Cyclic voltammograms were simulated using DigiSim software for reaction mechanisms involving multiple electron transfer steps coupled to proton transfer. Specifically, the overall reaction mechanism of the form O+2e(-)+2H(+) right harpoon over left harpoon RH(2) was used to simulate experimental reduction potentials as a function of pH. The pH-dependent reduction potentials reported in the literature for flavodoxin and free flavin adenine dinucleotide were simulated based on selected reduction potentials and acid dissociation constants. Relationships between reduction potentials and acid dissociation constants are presented to model n=1 and n=2 reaction mechanisms and one- and two-proton-coupled redox reactions. Experimental parameters used in the simulations were selected such that the electron and proton transfer reactions were not rate limiting, and therefore these simulated reactions involve thermodynamic coupling rather than concerted kinetic processes.

Redox properties of rubredoxin variants as a function of solvent composition and temperature: investigation of monopolar and dipolar interactions.

The role of solvent composition and temperature on equilibrium electron transfer in seven rubredoxin variants [ Clostridium pasteurianum ( Cp), V8D, V8R, V8A, V44A Cp, Pyrococcus furiosus ( Pf), and A44V Pf] were investigated to examine the role of both monopolar and dipolar interactions. The reduction potentials of all variants decreased as the polarity of the solvent decreased. The enthalpy and entropy associated with electron transfer were determined from temperature-controlled voltammetric studies. The entropic contribution [delta( Tdelta S degrees )] to the change in the reduction potential was larger for charged variants (V8D and V8R), while the enthalpic contribution [delta(-delta H degrees )] was larger for the other mutants. The large entropy change observed for monopolar variants is likely due to solvent reorganization that occurs between oxidation states. Entropic-enthalpic compensation phenomena, an observation that most proteins have an entropic term [delta( Tdelta S degrees )] and enthalpic term [delta(-delta H degrees )] with opposite signs, was observed. A correlation of the size of the amino acid side chain with delta E degrees ', delta(-delta H degrees ), and delta( Tdelta S degrees ) is also discussed.

Linear Free Energy Relationships in Dinuclear Compounds. 2. Inductive Redox Tuning via Remote Substituents in Quadruply Bonded Dimolybdenum Compounds.

Syntheses and characterizations are reported for dimolybdenum(II) compounds supported by the diarylformamidinate (ArNC(H)NAr(-)) ligand, where Ar is XC(6)H(4)(-), with X as p-OMe (1), H (2), m-OMe (3), p-Cl (4), m-Cl (5), m-CF(3) (6), p-COMe (7), p-CF(3) (8), or Ar is 3,4-Cl(2)C(6)H(3)(-) (9) or 3,5-Cl(2)C(6)H(3)(-) (10). The (quasi)reversible oxidation potentials measured for the Mo(2)(5+)/Mo(2)(4+) couple were found to correlate with the Hammett constant (sigma(X)) of the aryl substituents according to the following equation: DeltaE(1/2) = E(1/2)(X) - E(1/2)(H) = 87(8sigma(X)) mV. Molecular structure determinations of compounds 1, 2, 5, and 10 revealed an invariant core geometry around the Mo(2) center, with statistically identical Mo-Mo quadruple bond lengths of 2.0964(5), 2.0949[8], 2.0958(6), and 2.0965(5) Å, respectively. Magnetic anisotropies for compounds 1-10 estimated on the basis of (1)H NMR data were similar and unrelated to sigma(X). Similarity in UV-vis spectra was also found within the series, which, in conjunction with the features of both molecular structures and (1)H NMR spectra, was interpreted as the existence of a constant upper valence structure across the series. Results of Fenske-Hall calculations performed for several model compounds paralleled the experimental observations.