Modulation of flavocytochrome b2 intraprotein electron transfer via an interdomain hinge region.
The two domains of flavocytochrome b2 are connected by a typical hinge peptide. To probe the role of the hinge in modulating the efficiency of intraprotein electron transfer between these two domains, a number of mutant enzymes with truncated hinge regions were previously constructed and characterized [Sharp, Chapman and Reid (1996) Biochemistry 35, 891-899]. In the present study two mutant enzymes with elongated hinge regions have been constructed (HI3 and HI6) to further our understanding of the controlling influence of hinge length and primary structure 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 electron acceptor and from pre-steady-state experiments monitoring flavin reduction. However, the hinge insertions lowered the enzyme's effectiveness as a cytochrome c reductase. This effect results from a defect at the first interdomain electron-transfer step (FMNH2 --> haem electron transfer), where the rate constants for haem reduction in HI3 and HI6 were 50- and 100-fold lower than the corresponding value for the wild-type enzyme. Preservation of structural integrity within the hinge region is apparently essential for efficient intraprotein electron transfer.
Sharp
R E
RE
Edinburgh Centre for Molecular Recognition, University of Edinburgh, Scotland, U.K.
Chapman
S K
SK
Reid
G A
GA
D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D017931:DNA Primers; D004579:Electron Transport; D004926:Escherichia coli / Q000235:genetics; D005292:Ferricyanides / Q000378:metabolism; D005486:Flavin Mononucleotide / Q000378:metabolism; 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; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D017931:DNA Primers; D005292:Ferricyanides; D007773:Lactates; C007931:hexacyanoferrate III; D019344:Lactic Acid; D006418:Heme; D005486:Flavin Mononucleotide; D007770:L-Lactate Dehydrogenase; D042844:L-Lactate Dehydrogenase (Cytochrome)