Determination of the macroscopic formal potentials of cytochrome c3 of Desulfovibrio vulgaris, Miyazaki F, through the combined use of 1H NMR and an optically transparent thin-layer electrode cell

Abstract

The proton magnetic resonance ( 1H NMR) spectra of cytochrome c 3 from Desulfovibrio vulgaris, Miyazaki F, were obtained at various oxidation states. Five sets of heme methyl signals attributed to the five macroscopic oxidation states were classified. This classification provides firm evidence of the one-electron four-consecutive electron-transfer process of cytochrome c 3. The fraction of molecules in each macroscopic oxidation state was determined from the integrated intensity of the heme methyl signal, since the intermolecular electron exchange rate is slower and the intramolecular one is faster with respect to the NMR time scale. The equilibrium potential of cytochrome c 3 in an NMR tube was determined by measuring its visible absorption spectrum to correlate the absorption spectrum with the equlibrium potential. Four macroscopic formal potentials ( E o', i = 1, 2, 3 and 4) were determined directly from the fraction of molecules in each macroscopic oxidation state. The resulting formal potentials, E o' i , s, at 30° C in a 2H 2O solution were −260, −312, −327 and −369 mV vs. SHE (standard hydrogen electrode), respectively. These results agree quite well with those obtained by differential pulse polarography reported earlier.