Roles of the proximal heme thiolate ligand in cytochrome P450cam

K. Auclair, Pierre Moenne-Loccoz, P. R. Ortiz de Montellano

Research output: Contribution to journalArticle

101 Citations (Scopus)

Abstract

To examine the roles of the proximal thiolate iron ligand, the C357H mutant of P450cam (CYP101) was characterized by resonance Raman, UV, circular dichroism, and activity measurements. The C357H mutant must be reconstituted with hemin for activity to be observed. The reconstituted enzyme is a mixture of high and low spin species. Low temperature (10 °C), low enzyme concentration (1 μM), high camphor concentration (1 mM), and 5-50 mM buffer concentrations increase the high to low spin ratio, but under no conditions examined was the protein more than 60% high spin. The C357H mutant has a poorer Km for camphor (23 vs 2 μM) and a poorer Kd for putidaredoxin (50 vs 20 μM) than wild-type P450cam. The mutant also exhibits a greatly decreased camphor oxidation rate, elevated uncoupling rate, and much greater peroxidase activity. Electron transfer from putidaredoxin to the mutant is much slower than to the wild-type even though redox potential measurements show that the electron transfer remains thermodynamically favored. These experiments confirm that the thiolate ligand facilitates the O-O bond cleavage by P450 enzymes and also demonstrate that this ligand satisfies important roles in protein folding, substrate binding, and electron transfer.

Original languageEnglish (US)
Pages (from-to)4877-4885
Number of pages9
JournalJournal of the American Chemical Society
Volume123
Issue number21
DOIs
StatePublished - 2001

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Camphor 5-Monooxygenase
Camphor
Cytochromes
Heme
Ligands
Electrons
Proteins
Enzymes
Protein folding
Hemin
Protein Folding
Dichroism
Circular Dichroism
Cytochrome P-450 Enzyme System
Peroxidase
Oxidation-Reduction
Buffers
Iron
Oxidation
Temperature

ASJC Scopus subject areas

  • Chemistry(all)

Cite this

Roles of the proximal heme thiolate ligand in cytochrome P450cam . / Auclair, K.; Moenne-Loccoz, Pierre; Ortiz de Montellano, P. R.

In: Journal of the American Chemical Society, Vol. 123, No. 21, 2001, p. 4877-4885.

Research output: Contribution to journalArticle

Auclair, K. ; Moenne-Loccoz, Pierre ; Ortiz de Montellano, P. R. / Roles of the proximal heme thiolate ligand in cytochrome P450cam . In: Journal of the American Chemical Society. 2001 ; Vol. 123, No. 21. pp. 4877-4885.
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