Effect of Hydrogen-Bond Networks in Controlling Reduction Potentials in Desulfovibrio vulgaris (Hildenborough) Cytochrome c₃ Probed by Site-Specific Mutagenesis

Abstract

Cytochromes c 3 isolated from DesulfoVibrio spp. are periplasmic proteins that play a central role in energy transduction by coupling the transfer of electrons and protons from hydrogenase. Comparison between the oxidized and reduced structures of cytochrome c 3 isolated from DesulfoVibrio Vulgaris (Hildenborough) show that the residue threonine 24, located in the vicinity of heme III, reorients between these two states [Messias, A. C., Kastrau, D. H. W., Costa, H. S., LeGall, J., Turner, D. L., Santos, H., and Xavier, A. V. (1998) J. Mol. Biol. 281,. Threonine 24 was replaced with valine by sitedirected mutagenesis to elucidate its effect on the redox properties of the protein. The NMR spectra of the mutated protein are very similar to those of the wild type, showing that the general folding and heme core architecture are not affected by the mutation. However, thermodynamic analysis of the mutated cytochrome reveals a large alteration in the microscopic reduction potential of heme III (75 and 106 mV for the protonated forms of the fully reduced and oxidized states, respectively). The redox interactions involving this heme are also modified, while the remaining heme-heme interactions and the redoxBohr interactions are less strongly affected. Hence, the order of oxidation of the hemes in the mutated cytochrome is different from that in the wild type, and it has a higher overall affinity for electrons. This is consistent with the replacement of threonine 24 by valine preventing the formation of a network of hydrogen bonds, which stabilizes the oxidized state. The mutated protein is unable to perform a concerted two-electron step between the intermediate oxidation stages, 1 and 3, which can occur in the wild-type protein. Thus, replacing a single residue unbalances the global network of cooperativities tuned to control thermodynamically the directionality of the stepwise electron transfer and may affect the functionality of the protein.

Distance dependence of interactions between charged centres in proteins with common structural features

“…Since there is no apparent correlation between the interaction energy and the relative orientation of the haems, the charge was considered to be localised on the iron. Interactions involving acid–base centres were only considered for the cases of Dgc3, DvHc3 and DvMc3 in which the primary acid–base centre is unambiguously identified as the propionate 13 of haem I [20,24,25] and in this case the average position of the carboxylate oxygen atoms was considered as the location of the charge.…”

Metalloproteins Containing Cytochrome, Iron–Sulfur, or Copper Redox Centers

“…The reduction potentials of the hemes in cyts c 3 are also dependent on the pH, called the redox-Bohr effect, 340−342 due to the interactions of the heme propionates in the H-bonding network and/or electrostatic interactions with the residues in the vicinity. 341,343−345 …”

Desulfovibrio desulfuricans G20 Tetraheme Cytochrome Structure at 1.5Å and Cytochrome Interaction with Metal Complexes

“…Because molybdate was found to function as an electron acceptor from dithionite-reduced G20c 3 , and preliminary data from a site-directed mutant K14A of G20c 3 showed that molybdate could no longer oxidize this mutant protein (S. Miller & J.D.W., unpublished results), we infer that heme IV may be the primary site of electron exit from the protein, in agreement with other results. 12,20,41,44 Moreover, we predict that Lys56 is essential for reduction of molybdate, and possibly uranyl acetate, by G20c 3 . This prediction will be tested by mutagenesis studies.…”