Changing the heme ligation in flavocytochrome b(2): substitution of histidine-66 by cysteine

Mowat, Christopher G., Miles, Caroline S., Munro, Andrew W., Cheesman, Myles R., Quaroni, Luca G., Reid, Graeme A. and Chapman, Stephen K. (2000) Changing the heme ligation in flavocytochrome b(2): substitution of histidine-66 by cysteine. JBIC Journal of Biological Inorganic Chemistry, 5 (5). pp. 584-592. ISSN 0949-8257

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Substitution by cysteine of one of the heme iron axial ligands (His66) of flavocytochrome b(2) (L-lactate:cytochrome c oxidoreductase from Saccharomyces cerevisiae) has resulted in an enzyme (H66C-b(2)) which remains a competent L-lactate dehydrogenase (k(cat) 272+/-6 s(-1), L-lactate KM 0.60+/-0.06 mM, 25 degreesC, 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 v(4) band at 1345 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-b(2) heme is P450-like, Interestingly, EPR evidence also suggests that the second histidine heme-iron ligand (His43) is displaced in the mutant enzyme.

Item Type: Article
Faculty \ School: Faculty of Science > School of Chemistry
UEA Research Groups: Faculty of Science > Research Groups > Biophysical Chemistry (former - to 2017)
Faculty of Science > Research Groups > Chemistry of Life Processes
Faculty of Science > Research Centres > Centre for Molecular and Structural Biochemistry
Faculty of Science > Research Groups > Chemistry of Light and Energy
Depositing User: Rachel Smith
Date Deposited: 10 May 2011 11:14
Last Modified: 24 Oct 2022 03:55
DOI: 10.1007/s007750000141

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