DELETIONS IN THE INTERDOMAIN HINGE REGION OF FLAVOCYTOCHROME B(2) - EFFECTS ON INTRAPROTEIN ELECTRON-TRANSFER

The two distinct domains of flavocytochrome b(2) (L-lactate:cytochrome c oxidoreductase, EC 1.1.2.3) are connected by a typical hinge peptid e, To probe the importance of the structural integrity of the hinge re gion for efficient intraprotein electron transfer, three mutant enzyme s 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, an d H Delta 9 in which three, six, and nine amino acids, respectively, w ere deleted from the hinge region. Intraprotein electron transfer was investigated by steady-state and stopped-flow kinetic analyses. All th ree hinge-deletion enzymes remained good L-lactate dehydrogenases, as was evident from steady state experiments with ferricyanide as the ele ctron acceptor and from stopped-flow experiments monitoring flavin red uction. 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 b(2) is manifested at the first interdo main electron-transfer step (fully reduced FMN --> heme electron trans fer), where the rate of heme reduction is the same within experimental error as the steady-state rate of cytochrome c reduction. Thus, inter domain electron transfer is rate limiting in the case of these two hin ge-deletion enzymes compared to the wild-type enzyme, where alpha H ab straction from C-2 of L-lactate still contributes substantially to rat e limitation. The situation for H Delta 3 is more complicated, with mo re 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 b(2) catalytic cycle a nd show that complete structural integrity within the hinge region is essential for efficient interdomain communication.