Direct electrochemical studies have been performed on the dissimilatory 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) similar to -298 mV versus NHE by use of square-wave voltammetry with an edge pyrolytic graphite electrode (PGE) and redox inactive Cr(NH3)(6)(3+) promoter. The half-height peak width of 122 mV is in excellent agreement with the theoretical value of 126 mV expected for a reversible one-electron transfer with no coupled chemical reaction. Uptake of a second 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 mV versus NHE was measured with a Hg(1) pool electrode by controlled potential coulometry (CPC). The reduction potentials for the first and second redox couples of the [Fe4S4]-siroheme prosthetic center in the assimilatory enzyme have been determined as E(1) degrees' similar to -21 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 for 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 controlled reversible heterogeneous electron transfer is observed for mu M enzyme concentrations of either enzyme. For all signals observed in SWV the peak current (i(p)) is proportional to the square-root of the frequency (v) of the applied potential, indicative of diffusion control 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 have been determined from variable temperature experiments as follows (pH 7.0 and 25 degrees C): DSiR, Delta H degrees' -3.0 kcal mol(-1), Delta S degrees' -31.3 eu; ASiR, Delta H degrees' (siroheme) -11.8 kcal mol(-1), Delta S degrees' (siroheme) -36.4 eu; Delta H degrees' (cluster) -4.5 kcal mol(-1), Delta S degrees' (cluster) -34.7 eu. Comparison is made with the redox thermodynamic parameters of cyt c (horse), myoglobin, and high potential iron protein. Systematic pH-titration studies provide evidence for coordination of an ionizable ligand to the prosthetic redox center, which is likely to be a bridging sulfide. For DSiR the electrochemical response for the first redox couple remains a single reversible peak over the entire pH range with a half-height peak width expected for one-electron exchange. The pH titration results support direct coupling of the siroheme and Fe4S4 cluster by the bridging ligand. The siroheme redox couple of ASiR shows an additional response in the pH titration curve (ascribed to an axial histidine residue) that is superimposed on the major broad pH-response from the enzyme. The detailed pH-dependence of both enthalpic and entropic parameters has been examined, and implications for active site chemistry are discussed. For DSiR, both Delta H degrees and Delta S degrees terms show no clear pH-dependence over the range from 3 to 10; however, for ASiR both the entropic and enthalpic components show a pH-dependence with an estimated pK(a)(ox) similar to 5.8 and pK(a)(red) of similar to 7.6. This is particularly pronounced for the siroheme redox couple and is proposed to originate from release of the axial histidine residue following reduction of the enzyme.
