JBIC Journal of Biological Inorganic Chemistry

, Volume 5, Issue 5, pp 584–592

Changing the heme ligation in flavocytochrome b2: substitution of histidine-66 by cysteine

  • Christopher G. Mowat
  • Caroline S. Miles
  • Andrew W. Munro
  • Myles R. Cheesman
  • Luca G. Quaroni
  • Graeme A. Reid
  • Stephen K. Chapman
Original Article

DOI: 10.1007/s007750000141

Cite this article as:
Mowat, C., Miles, C., Munro, A. et al. JBIC (2000) 5: 584. doi:10.1007/s007750000141

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 °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 ν4 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-b2 heme is P450-like. Interestingly, EPR evidence also suggests that the second histidine heme-iron ligand (His43) is displaced in the mutant enzyme.

Flavocytochrome l-Lactate dehydrogenase Heme-iron ligation Site-directed mutagenesis

Copyright information

© SBIC 2000

Authors and Affiliations

  • Christopher G. Mowat
    • 1
  • Caroline S. Miles
    • 2
  • Andrew W. Munro
    • 3
  • Myles R. Cheesman
    • 4
  • Luca G. Quaroni
    • 5
  • Graeme A. Reid
    • 2
  • Stephen K. Chapman
    • 1
  1. 1.Department of Chemistry, University of Edinburgh, Kings Buildings, West Mains Road, Edinburgh, EH9 3JJ, UK
  2. 2.Institute of Cell and Molecular Biology, University of Edinburgh, Kings Buildings, Mayfield Road, Edinburgh, EH9 3JR, UK
  3. 3.Department of Pure and Applied Chemistry, University of Strathclyde, The Royal College, George Street, Glasgow, G1 1XL, UK
  4. 4.School of Chemical Sciences, University of East Anglia, Norwich, NR4 7TJ, UK
  5. 5.Department of Chemistry, University of Minnesota, 400 Smith Hall, 207 Pleasant Street, Minneapolis, MN 55455, USA