4-Cyanopyridine, a versatile spectroscopic probe for cytochrome P450BM3

TWB Ost, JP Clark, LJ Yellowlees, S Daff, SK Chapman*, J L R Anderson

*Corresponding author for this work

Research output: Contribution to journalArticle (Academic Journal)peer-review

14 Citations (Scopus)

Abstract

The nitrogenous pi-acceptor ligand 4-cyanopyridine (4CNPy) exhibits reversible ligation to ferrous heme in the flavocytochrome P450 BM3 (K-d=1.8 muM for wild type P450 BM3) via its pyridine ring nitrogen. The reduced P450-4CNPy adduct displays unusual spectral properties that provide a useful spectroscopic handle to probe particular aspects of this P450. 4CNPy is competitively displaced upon substrate binding, allowing a convenient route to the determination of substrate dissociation constants for ferrous P450 highlighting an increase in P450 substrate affinity on heme reduction. For wild type P450 BM3, K-d(red)(laurate)=82.4 muM (cf. K-d(ox)=364 muM). In addition, an unusual spectral feature in the red region of the absorption spectrum of the reduced P450-4CNPy adduct is observed that can be assigned as a metal-to-ligand charge transfer (MLCT). It was discovered that the energy of this MLCT varies linearly with respect to the P450 heme reduction potential. By studying the energy of this MLCT for a series of BM3 active site mutants with differing reduction potential (E-m), the relationship E-MLCT=(3.53 x E-m)+17,005 cm(-1) was derived. The use of this ligand thus provides a quick and accurate method for predicting the heme reduction potentials of a series of P450 BM3 mutations using visible spectroscopy, without the requirement for redox potentiometry.

Original languageEnglish
Pages (from-to)48876-48882
Number of pages7
JournalJournal of Biological Chemistry
Volume279
Issue number47
DOIs
Publication statusPublished - 19 Nov 2004

Keywords

  • PSEUDOMONAS-PUTIDA CYTOCHROME-P-450
  • FLAVOCYTOCHROME P-450 BM3
  • CRYSTAL-STRUCTURE
  • ELECTRON-TRANSFER
  • LIVER MICROSOMES
  • HEME
  • SPIN
  • BINDING
  • SUBSTRATE
  • COMPLEXES

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