STRUCTURAL AND KINETIC-STUDIES OF THE Y73E MUTANT OF OCTAHEME CYTOCHROME C(3) (M-R=26 000) FROM DESULFOVIBRIO-DESULFURICANS NORWAY

A combination of structural, kinetic, and interaction experiments has been used to study the role of a highly conserved aromatic residue, Ty r73, parallel to the sixth heme axial ligand of heme 4 in multiheme cy tochrome c(3) (M-r, = 26 000), also called cytochrome cc(3) or octahem e cytochrome, from Desulfovibrio desulfuricans Norway. This residue is expected to be involved in intermolecular electron transfer and prote in-protein interaction, since heme 4 is described to be the interactio n site between physiological partners, The kinetic experiments show th at the Y73E replacement provokes no significant change in the electron -transfer reaction with the physiological partner, the [NiFeSe] hydrog enase, but that the protein-protein interaction between cytochrome c(3 ) (M-r = 26 000) and hydrogenase is strongly affected by the mutation. The aromatic residue does not play a role in maintaining the axial he me ligand in a particular orientation, since the mutation did not affe ct The orientation of histidine 77, the sixth axial Ligand of heme 4. The structural analysis by X-ray crystallography clearly shows that a rearrangement of the charged residues in the Vicinity of the mutation site is responsible for the change in protein-protein interaction, whi ch is of an electrostatic nature. Lys22 and Arg66, residues which are located at the interacting,a surface, are twisted toward the mutated p osition Glu73 in order to compensate for the negative charge and there fore are no longer accessible for the docking with a physiological par tner, Tyr73 has instead a structural function and probably a role in m aintaining the hydrophobic environment of the heme 4 cavity rather tha n a function in the intermolecular electron transfer with the physiolo gical partners.