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Reference: Mowat CG, et al. (2000) Changing the heme ligation in flavocytochrome b2: substitution of histidine-66 by cysteine. J Biol Inorg Chem 5(5):584-92

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Abstract


Substitution by cysteine of one of the heme iron axial ligands (His66) of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase from Saccharomyces cerevisiae) has resulted in an enzyme (H66C-b2) which remains a competent L-lactate dehydrogenase (kcat 272+/-6 s(-1), L-lactate KM 0.60+/-0.06 mM, 25 degrees C, I 0.10, Tris-HCl, pH 7.5) but which has no cytochrome c reductase activity. As a result of the mutation, the reduction potential of the heme was found to be -265+5 mV, over 240 mV more negative than that of the wild-type enzyme, and therefore unable to be reduced by L-lactate. Surface-enhanced resonance Raman spectroscopy indicates similarities between the heme of H66C-b2 and those of cytochromes P450, with a nu4 band at 1,345 cm(-1) which is indicative of cysteine heme-iron ligation. In addition, EPR spectroscopy yields g-values at 2.33, 2.22 and 1.94, typical of low-spin ferric cytochromes P450, optical spectra show features between 600 and 900 nm which are characteristic of sulfur coordination of the heme iron, and MCD spectroscopy shows a blue-shifted NIR CT band relative to the wild-type, implying that the H66C-b2 heme is P450-like. Interestingly, EPR evidence also suggests that the second histidine heme-iron ligand (His43) is displaced in the mutant enzyme.

Reference Type
Journal Article | Research Support, Non-U.S. Gov't
Authors
Mowat CG, Miles CS, Munro AW, Cheesman MR, Quaroni LG, Reid GA, Chapman SK
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