A proton-NMR investigation of the fully reduced cytochrome c(7) from Desulfuromonas acetoxidans - Comparison between the reduced and the oxidized forms

The solution structure via H-1 NMR of the fully reduced form of cytochrome c(7) has been obtained. The protein sample was kept reduced by addition of catalytic amounts of Desulfovibrio gigas iron hydrogenase in H-2 atmosphere after it had been checked that the presence of the hydrogenase did not aff ect the NMR spectrum. A final family of 35 conformers with rmsd values with respect to the mean structure of 8.7 +/- 1.5 nm and 12.4 +/- 1.3 nm for th e backbone and heavy atoms, respectively, was obtained. A highly disordered loop involving residues 54-61 is present. If this loop is ignored, the rms d values are 6.2 +/- 1.1 nm and 10.2 +/- 1.0 nm for the backbone and heavy atoms, respectively, which represent a reasonable resolution. The structure was analyzed and compared with the already available structur e of the fully oxidized protein. Within the indetermination of the two solu tion structures, the result for the two redox forms is quite similar, confi rming the special structural features of the three-heme cluster. A useful c omparison can be made with the available crystal structures of cytochromes c(3), which appear to be highly homologous except for the presence of a fur ther heme. Finally, an analysis of the factors affecting the reduction pote ntials of the heme irons was performed, revealing the importance of net cha rges in differentiating the reduction potential when the other parameters a re kept constant.