Electron transfer in tetrahemic cytochromes c3: spectroelectrochemical evidence for a conformational change triggered by heme IV reduction.

Electron transfer in tetrahemic cytochromes c3 from Desulfovibrio vulgaris Hildenborough (D.v.H.) and Desulfovibrio desulfuricans Norway (D.d.N.) strains has been investigated by thin layer spectroelectrochemistry with visible absorption, CD, and resonance Raman (RR) monitoring. The observed splitting of the isosbestic point in the Soret absorption band indicates that the electron transfer from the (FeIII)4 state to the (FeII)4 state proceeds via an intermediate species, which corresponds to 25 and 50% reduction for the D.v.H. cyt.c3 and the D.d.N. cyt.c3, respectively. For the latter, a specific CD signal is observed at half-reduction. RR monitoring of the redox process does not reveal multiple splitting of the high-frequency RR bands, at variance with previously published results on the enzymatic reduction of cyt.c3 from Desulfovibrio vulgaris Miyazaki, a cytochrome highly homologous to D.v.H. cyt.c3 [Verma, A.L., Kimura, A., Nakamura, A., Yagi, T., Inoguchi, H., & Kitagawa, T. (1988) J. Am. Chem. Soc. 110, 6617-6623]. The low-frequency RR spectra of the intermediate species differ significantly from the ones calculated from a linear combination of the all-ferric and all-ferrous states, for the same reduction ratio. Frequency shifts of the bending modes of the cysteine and propionate heme substituents are observed, as well as changes specific to each cytochrome; most notable is the activation of two torsional modes in the case of D.d.N. cyt.c3. Comparison of the results obtained for the two cytochromes leads to the conclusion that reduction of heme IV triggers the observed conformational change. This conclusion is supported by the spectroelectrochemical investigation of the mutant D.v.H. cyt.c3 H25M, in which the sixth ligand of heme III, histidine, is replaced by a methionine.