Deletions in the interdomain hinge region of flavocytochrome b2: effects on intraprotein electron transfer.
The two distinct domains of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase, EC 1.1.2.3) are connected by a typical hinge peptide. To probe the importance of the structural integrity of the hinge region for efficient intraprotein electron transfer, three mutant enzymes have been constructed: H delta 3 [Sharp, R. E., White, P., Chapman, S. K., & Reid, G. A. (1994) Biochemistry 33, 5115-5120], H delta 6, and H delta 9 in which three, six, and nine amino acids, respectively, were deleted from the hinge region. Intraprotein electron transfer was investigated by steady-state and stopped-flow kinetic analyses. All three hinge-deletion enzymes remained good L-lactate dehydrogenases, as was evident from steady-state experiments with ferricyanide as the electron acceptor and from stopped-flow experiments monitoring flavin reduction. The global effect of these deletions is to lower the enzyme's effectiveness as a cytochrome c reductase. This property of H delta 6 and H delta 9 flavocytochromes b2 is manifested at the first interdomain electron-transfer step (fully reduced FMN-->heme electron transfer), where the rate of heme reduction is the same within experimental error as the steady-state rate of cytochrome c reduction. Thus, interdomain electron transfer is rate limiting in the case of these two hinge-deletion enzymes compared to the wild-type enzyme, where alpha H abstraction from C-2 of L-lactate still contributes substantially to rate limitation. The situation for H delta 3 is more complicated, with more than one interdomain electron-transfer step being affected. Kinetic data, along with the measured deuterium kinetic isotope effects, are discussed in the context of the flavocytochrome b2 catalytic cycle and show that complete structural integrity within the hinge region is essential for efficient interdomain communication.
Institute of Cell and Molecular Biology, Edinburgh Centre for Molecular Recognition, University of Edinburgh, Scotland.
R E
RE
Sharp
S K
SK
Chapman
G A
GA
Reid
D000595:Amino Acid Sequence; D006418:Heme / Q000378:metabolism; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D042844:L-Lactate Dehydrogenase (Cytochrome); D007773:Lactates / Q000378:metabolism; D019344:Lactic Acid; D008969:Molecular Sequence Data; D009154:Mutation; D010084:Oxidation-Reduction
D007773:Lactates; D019344:Lactic Acid; D006418:Heme; D007770:L-Lactate Dehydrogenase; D042844:L-Lactate Dehydrogenase (Cytochrome)