Changing the heme ligation in flavocytochrome b2: substitution of histidine-66 by cysteine.
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.
Mowat
C G
CG
Department of Chemistry, University of Edinburgh, UK.
Miles
C S
CS
Munro
A W
AW
Cheesman
M R
MR
Quaroni
L G
LG
Reid
G A
GA
Chapman
S K
SK
D019943:Amino Acid Substitution / Q000235:genetics*; D002942:Circular Dichroism; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D005486:Flavin Mononucleotide / Q000378:metabolism; D006418:Heme / Q000378:metabolism*; D006639:Histidine / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D042844:L-Lactate Dehydrogenase (Cytochrome); D009154:Mutation / Q000235:genetics; D010084:Oxidation-Reduction; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D013053:Spectrophotometry; D013059:Spectrum Analysis, Raman
D006418:Heme; D006639:Histidine; D005486:Flavin Mononucleotide; D007770:L-Lactate Dehydrogenase; D042844:L-Lactate Dehydrogenase (Cytochrome); D003545:Cysteine