Substitution of a haem-iron axial ligand in flavocytochrome b2.
The importance of haem-iron axial coordination in flavocytochrome b2 (L-lactate: cytochrome-c oxidoreductase) has been examined by replacing one of the ligating histidines, His-43, with methionine. The His-43-->Met mutation (H43M) results in a distinct colour change from red in the wild-type enzyme to green in the mutant enzyme. The electronic absorption spectrum indicates that only approx. 5% of the haem binding sites are occupied. There is no evidence of any absorption band at 695 nm (characteristic of methionine ligation) suggesting that methionine does not act as an axial ligand in the mutant enzyme. The H43M-mutant enzyme shows a band around 640-650 nm which is usually associated with high-spin ferric-haem proteins, either five coordinate or with a weak-field ligand in the sixth position. The EPR spectrum of the H43M-enzyme at 7 K shows a g-value near 6.0, indicating that the haem-iron is high-spin in contrast to its low-spin state in the wild-type enzyme. The His-43-->Met mutation has only a small effect on the lactate dehydrogenase activity of the enzyme as measured with ferricyanide as external electron acceptor, but greatly reduces its cytochrome-c reductase activity.
Miles
C S
CS
Edinburgh Centre for Molecular Recognition, Department of Chemistry, University of Edinburgh, UK.
Manson
F D
FD
Reid
G A
GA
Chapman
S K
SK
D001483:Base Sequence; D015786:Cytochromes b5 / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology; D006418:Heme / Q000737:chemistry*; D006639:Histidine; D007501:Iron / Q000737:chemistry*; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000737:chemistry*; D042844:L-Lactate Dehydrogenase (Cytochrome); D008715:Methionine; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D013057:Spectrum Analysis
D006418:Heme; D006639:Histidine; D015786:Cytochromes b5; D008715:Methionine; D007501:Iron; D007770:L-Lactate Dehydrogenase; D042844:L-Lactate Dehydrogenase (Cytochrome)