Cytochrome c3 isolated from a sulfate-reducing bacterium, Desulfovibrio vulgaris Miyazaki F, is a tetraheme protein. Its physiological partner, [NiFe] hydrogenase, catalyzes the reversible oxidoreduction of molecular hydrogen. To elucidate the mechanism of electron transfer between cytochrome c3 and [NiFe] hydrogenase, the transient complex formation by these proteins was investigated by means of NMR. All NH signals of uniformly 15N-labeled ferric cytochrome c3 except N-terminus, Pro, and Gly73 were assigned. 1H-15N HSQC spectra were recorded for 15N-labeled ferric and ferrous cytochrome c3, in the absence and presence of hydrogenase. Chemical shift perturbations were observed in the region around heme 4 in both oxidation states. Additionally, the region between hemes 1 and 3 in ferrous cytochrome c3 was affected in the presence of hydrogenase, suggesting that the mode of interaction is different in each redox state. Heme 3 is probably the electron gate for ferrous cytochrome c3. To investigate the transient complex of cytochrome c3 and hydrogenase in detail, modeling of the complex was performed for the oxidized proteins using a docking program, ZDOCK 2.3, and NMR data. Furthermore, the roles of lysine residues of cytochrome c3 in the interaction with hydrogenase were investigated by site-directed mutagenesis. When the lysine residues around heme 4 were replaced by an uncharged residue, methionine, one by one, the Km of the electron-transfer kinetics increased. The results showed that the positive charges of Lys60, Lys72, Lys95, and Lys101 around heme 4 are important for formation of the transient complex with [NiFe] hydrogenase in the initial stage of the cytochrome c3 reduction. This finding is consistent with the most possible structure of the transient complex obtained by modeling.

Carbon monoxide (CO) has been identified as another bioactive molecule like NO. Binding of CO to a tetraheme cytochrome c(3) (cyt c(3)) was investigated using visible absorption spectroscopy, circular dichroism (CD), and NMR. CO was found to bind to the four hemes in different manners. CD spectra, however, indicated that only single-site CO binding can keep the protein intact. The K(d) for the single-site binding was 8.0 microM, which is a typical value for a CO sensor protein. Furthermore, NMR spectra of uniformly (15)N-labeled and specifically [(15)N]His-labeled proteins have provided evidence that CO specifically binds to the sixth coordination site of heme 2 via single-site binding. The CO-bound cyt c(3) could conduct redox reactions. In light of triheme cytochrome c(7), the CO-bound cyt c(3) may work as an electron transporter. It was reported for sulfate-reducing bacteria that CO can be used as an energy source and CO cycling is operating like H(2) cycling. Therefore, the CO-bound cyt c(3) may play a role in maintaining electron transport pathways on accumulation of toxic CO for its utilization.

Complicated pH-properties of the tetraheme cytochrome c3 (cyt c3) from Desulfovibrio vulgaris Miyazaki F (DvMF) were examined by the pH titrations of 1H-15N HSQC spectra in the ferric and ferrous states. The redox-linked pKa shift for the propionate group at C13 of heme 1 was observed as the changes of the NH signals around it. This pKa shift is consistent with the redox-linked conformational alteration responsible for the cooperative reduction between hemes 1 and 2. On the other hand, large chemical shift changes caused by the protonation/deprotonation of Glu41 and/or Asp42, and His67 were redox-independent. Nevertheless, these charged residues affect the redox properties of the four hemes. Furthermore, one of interesting charged residues, Glu41, was studied by site-directed mutagenesis. E41K mutation increased the microscopic redox potentials of heme 1 by 46 and 34 mV, and heme 2 by 35 and 30 mV at the first and last reduction steps, respectively. Although global folding in the crystal structure of E41K cyt c3 is similar to that of wild type, local change was observed in 1H NMR spectrum. Glu41 is important to keep the stable conformation in the region between hemes 1 and 2, controlling the redox properties of DvMF cyt c3. In contrast, the kinetic parameters for electron transfer from DvMF [NiFe] hydrogenase were not influenced by E41K mutation. This suggests that the region between hemes 1 and 2 is not involved in the interaction with [NiFe] hydrogenase, and it supports the idea that heme 4 is the exclusive entrance gate to accept the electron in the initial reduction stage.