LOCATION OF A CYTOCHROME-C BINDING-SITE ON THE SURFACE OF FLAVOCYTOCHROME B(2)

Saccharomyces cerevisiae flavocytochrome b(2) couples the oxidation of L-lactate to the reduction of cytochrome c. The second-order rate con stant for cytochrome c reduction by flavocytochrome b(2) depends on th e rate of complex formation and is sensitive to ionic strength, Mutati ons in the heme domain of flavocytochrome b(2) (Glu63-->Lys, Asp72-->L ps and the double mutation GluG3-->Lys:Asp72-->Lys) have significant e ffects on the reaction with cytochrome c, implicating these residues i n complex formation. This kinetic information has been used to guide m olecular modelling studies, which are consistent with there being no o ne single best-configuration. Rather, there is a set of possible compl exes in which the docking-face of cytochrome c can approach flavocytoc hrome b(2) in a variety of orientations, Four cytochromes c can be acc ommodated on the flavocytochrome b(2) tetramer, with each cytochrome c forming interactions with only one flavocytochrome b(2) subunit. All the models involve residues 72 and 63 on flavocytochrome b(2) but in a ddition predict that GIu237 may also be important for complex formatio n. These acidic residues interact with the basic residues 13, 27 and 7 9 on cytochrome c, Through this triangle of interactions runs a possib le sigma-tunnelling pathway for electron transfer. This pathway starts with the imidazole ring of His66 (a ligand to the heme-iron of flavoc ytochrome b(2)) and ends with the ring of Pro68, which is in van der W aals contact with the cytochrome c heme, In total, the edge-to-edge '' through space'' distance from the imidazole ring of His66 to the C3C p yrrole ring of cytochrome c is 13.1 Angstrom.