Role of the interdomain hinge of flavocytochrome b2 in intra- and inter-protein electron transfer.
The two distinct domains of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase, EC 1.1.2.3) are connected by a hinge peptide. Kinetics experiments [White, P., Manson, F. D. C., Brunt, C. E., Chapman, S. K., & Reid, GA. (1993) Biochem. J. 291, 89-94] have illustrated the importance for efficient interdomain electron transfer of maintaining the structural integrity of the hinge. To probe the role of the hinge in a more subtle manner, we have constructed a mutant enzyme, H delta 3, which has a three amino acid deletion in the hinge region. Intra- and inter-protein electron transfer within H delta 3 flavocytochrome b2 and the H delta 3:cytochrome c redox complex was investigated by steady-state and stopped-flow kinetics analysis. The H delta 3 mutant enzyme remains a good L-lactate dehydrogenase, as is evident from steady-state experiments with ferricyanide as electron acceptor (40% less active than wild-type enzyme) and stopped-flow experiments monitoring flavin reduction (15% less active than wild-type enzyme). The global effect of the deletion is to lower the enzyme's effectiveness as a cytochrome c reductase. This property of the H delta 3 enzyme is manifested at two electron-transfer steps on the catalytic cycle of flavocytochrome b2. First, the rate of heme reduction has fallen 5-fold in H delta 3 compared with the wild-type enzyme (from 445 to 91 s-1), due to poor interdomain electron transfer from flavin to heme.(ABSTRACT TRUNCATED AT 250 WORDS)
Sharp
R E
RE
Institute of Cell and Molecular Biology, Edinburgh Centre for Molecular Recognition, University of Edinburgh, Scotland, U.K.
White
P
P
Chapman
S K
SK
Reid
G A
GA
D000595:Amino Acid Sequence; D001483:Base Sequence; D003196:Computer Graphics; D004579:Electron Transport; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D042844:L-Lactate Dehydrogenase (Cytochrome); D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D007770:L-Lactate Dehydrogenase; D042844:L-Lactate Dehydrogenase (Cytochrome)