Proximal cysteine residue is essential for the enzymatic activities of cytochrome p450cam

Shiro Yoshioka, Satoshi Takahashi, Hiroshi Hori, Koichiro Ishimori, Isao Morishima

Research output: Contribution to journalArticle

45 Citations (Scopus)

Abstract

To investigate the functional and structural roles of the proximal thiolate ligand in cytochrome P450cam, we prepared the C357H mutant of the enzyme in which the axial cysteine residue (Cys357) was replaced with a histidine residue. We obtained the unstable C357H mutant by developing a new preparation procedure involving in vitro folding of P450cam from the inclusion bodies. The C357H mutant in the ferrous-CO form exhibited the Soret peak at 420 nm and the Fe-CO stretching line at 498 cm-1, indicating a neutral histidine residue as the axial ligand. However, another internal ligand is coordinated to the heme iron as the sixth ligand in the ferric and ferrous forms of the C357H mutant, suggesting the collapse of the substrate-binding site. The C357H mutant showed no catalytic activity for camphor hydroxylation and the reduced heterolytic/homolytic ratio of the O-O bond scission in the reaction with cumene hydroperoxide. The present observations indicate that the thiolate coordination in P450cam is important for the construction of the heme pocket and the heterolysis of the O-O bond.

Original languageEnglish
Pages (from-to)252-259
Number of pages8
JournalEuropean Journal of Biochemistry
Volume268
Issue number2
DOIs
Publication statusPublished - 2001

Keywords

  • Axial push effect
  • CPO
  • Oxygen activation
  • P420
  • P450

ASJC Scopus subject areas

  • Biochemistry

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