Structural basis for the network of functional cooperativities in cytochrome c(3) from Desulfovibrio gigas: Solution structures of the oxidised and reduced states

Cytochrome c(3) is a 14 kDa tetrahaem protein that plays a central role in the bioenergetic metabolism of Desulfovibrio spp. This involves an energy t ransduction mechanism made possible by a complex network of functional coop erativities between redox and redox/protolytic centres (the redox-Bohr effe ct), which enables cytochrome c(3) to work as a proton activator. The three -dimensional structures of the oxidised and reduced Desulfovibrio gigas cyt ochrome c(3) in solution were solved using 2D H-1-NMR data. The reduced pro tein structures were calculated using INDYANA, an extended version of DYANA that allows automatic calibration of NOE data. The oxidised protein struct ure, which includes four paramagnetic centres, was solved using the program PARADYANA, which also includes the structural paramagnetic parameters. In this case, initial structures were used to correct the upper and lower volu me restraints for paramagnetic leakage, and angle restraints derived from C -13 Fermi contact shifts of haem moiety substituents were used for the axia l histidine ligands. Despite the reduction of the NOE intensities by parama gnetic relaxation, the final family of structures is of similar precision a nd accuracy to that obtained for the reduced form. Comparison of the two st ructures shows that, although the global folds of the two families of struc tures are similar, significant localised differences occur upon change of r edox state, some of which could not be detected by comparison with the X-ra y structure of the oxidised state: (1) there is a redox-linked concerted re arrangement of Lys80 and Lys90 that results in the stabilisation of haem mo ieties II and III when both molecules are oxidised or both are reduced, in agreement with the previously measured positive redox cooperativity between these two haem moieties. This cooperativity regulates electron transfer, e nabling a two-electron step adapted to the function of cytochromes c(3) as the coupling partner of hydrogenase; and (2) the movement of haem I propion ate 13 towards the interior of the protein upon reduction explains the posi tive redox-Bohr effect, establishing the structural basis for the redox-lin ked proton activation mechanism necessary for energy conservation, driving Am synthesis. (C) 2000 Academic Press.