Deletions in the Interdomain Hinge Region of Flavocytochrome b2: Effects on Intraprotein Electron Transfer
- 1 January 1996
- journal article
- research article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 35 (3), 891-899
- https://doi.org/10.1021/bi950457z
Abstract
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: HΔ3 [Sharp, R. E., White, P., Chapman, S. K., & Reid, G. A. (1994) Biochemistry 33, 5115−5120], HΔ6, and HΔ9 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 HΔ6 and HΔ9 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 αH abstraction from C-2 of l-lactate still contributes substantially to rate limitation. The situation for HΔ3 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.Keywords
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