DIRECT REVERSIBLE PROTEIN ELECTROCHEMISTRY AT A PYROLYTIC-GRAPHITE ELECTRODE - CHARACTERIZATION OF THE REDOX THERMODYNAMICS OF THE FE4S4 SIROHEME PROSTHETIC CENTER IN THE HEXAMERIC DISSIMILATORY SULFITE REDUCTASE AND THE MONOMERIC ASSIMILATORY SULFITE REDUCTASE FROM DESULFOVIBRIO-VULGARIS (HILDENBOROUGH) - SYSTEMATIC PH TITRATION EXPERIMENTS AND IMPLICATIONS FOR ACTIVE-SITE CHEMISTRY

Direct electrochemical studies have been performed on the dissimilator y hexameric sulfite reductase (DSiR, M(r) similar to 200 000) and the assimilatory monomeric sulfite reductase (ASiR, M(r) similar to 23 500 ) from Desulfovibrio vulgaris (Hildenborough). The reduction potential for the first redox couple of the [Fe4S4]-siroheme prosthetic center in DSiR has been determined as E degrees' (25 degrees C, pH 7.5) simil ar to -298 mV versus NHE by use of square-wave voltammetry with an edg e pyrolytic graphite electrode (PGE) and redox inactive Cr(NH3)(6)(3+) promoter. The half-height peak width of 122 mV is in excellent agreem ent with the theoretical value of 126 mV expected for a reversible one -electron transfer with no coupled chemical reaction. Uptake of a seco nd electron occurs at a reduction potential that is too negative to be detected over the range allowed while using the Cr(NH3)(6)(3+) redox promoter. The second reduction potential, E degrees' similar to -620 m V versus NHE was measured with a Hg(1) pool electrode by controlled po tential coulometry (CPC). The reduction potentials for the first and s econd redox couples of the [Fe4S4]-siroheme prosthetic center in the a ssimilatory enzyme have been determined as E(1) degrees' similar to -2 1 mV (siroheme) and E(2) degrees' similar to -303 mV (cluster) versus NHE at pH 7.5 and 25 degrees C. The half-height peak width of 134 mV f or the first redox couple (siroheme) is again in close agreement with the theoretical value of 126 mV expected for a reversible redox couple involving one-electron transfer; however, the half-height peak width for the second redox couple (Fe4S4 cluster) is only 84 mV. Diffusion c ontrolled reversible heterogeneous electron transfer is observed for m u M enzyme concentrations of either enzyme. For all signals observed i n SWV the peak current (i(p)) is proportional to the square-root of th e frequency (v) of the applied potential, indicative of diffusion cont rol and rapid equilibrium conditions. The variation of peak current (i (p)) with the amplitude of the square wave pulse (E(p)) has also been examined. Enthalpic and entropic contributions to E degrees' values ha ve been determined from variable temperature experiments as follows (p H 7.0 and 25 degrees C): DSiR, Delta H degrees' -3.0 kcal mol(-1), Del ta S degrees' -31.3 eu; ASiR, Delta H degrees' (siroheme) -11.8 kcal m ol(-1), Delta S degrees' (siroheme) -36.4 eu; Delta H degrees' (cluste r) -4.5 kcal mol(-1), Delta S degrees' (cluster) -34.7 eu. Comparison is made with the redox thermodynamic parameters of cyt c (horse), myog lobin, and high potential iron protein. Systematic pH-titration studie s provide evidence for coordination of an ionizable ligand to the pros thetic redox center, which is likely to be a bridging sulfide. For DSi R the electrochemical response for the first redox couple remains a si ngle reversible peak over the entire pH range with a half-height peak width expected for one-electron exchange. The pH titration results sup port direct coupling of the siroheme and Fe4S4 cluster by the bridging ligand. The siroheme redox couple of ASiR shows an additional respons e in the pH titration curve (ascribed to an axial histidine residue) t hat is superimposed on the major broad pH-response from the enzyme. Th e detailed pH-dependence of both enthalpic and entropic parameters has been examined, and implications for active site chemistry are discuss ed. For DSiR, both Delta H degrees and Delta S degrees terms show no c lear pH-dependence over the range from 3 to 10; however, for ASiR both the entropic and enthalpic components show a pH-dependence with an es timated pK(a)(ox) similar to 5.8 and pK(a)(red) of similar to 7.6. Thi s is particularly pronounced for the siroheme redox couple and is prop osed to originate from release of the axial histidine residue followin g reduction of the enzyme.