MODULATION OF FLAVOCYTOCHROME B(2) INTRAPROTEIN ELECTRON-TRANSFER VIAAN INTERDOMAIN HINGE REGION

The two domains of flavocytochrome b(2) are connected by a typical hin ge peptide, To probe the role of the hinge in modulating the efficienc y of intraprotein electron transfer between these two domains, a numbe r of mutant enzymes with truncated hinge regions were previously const ructed and characterized [Sharp, Chapman and Reid (1996) Biochemistry 35, 891-899]. In the present study two mutant enzymes with elongated h inge regions have been constructed (HI3 and HI6) to further our unders tanding of the controlling influence of hinge length and primary struc ture on intraprotein electron transfer. Modification of the hinge had little effect on the lactate dehydrogenase activity of the enzyme, as was evident from steady-state experiments using ferricyanide as electr on acceptor and from pre-steady-state experiments monitoring flavin re duction. However, the hinge insertions lowered the enzyme's effectiven ess as a cytochrome c reductase, This effect results from a defect at the first interdomain electron-transfer step (FMNH(2) --> haem electro n transfer), where the rate constants for haem reduction in Hn and HI6 were 50- and 100-fold lower than the corresponding value for the wild -type enzyme. Preservation of structural integrity within the hinge re gion is apparently essential for efficient intraprotein electron trans fer.