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J. Biol. Chem., Vol. 279, Issue 47, 48876-48882, November 19, 2004

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4-Cyanopyridine, a Versatile Spectroscopic Probe for Cytochrome P450 BM3^*

Tobias W. B. Ost, Jonathan P. Clark, J. L. Ross Anderson, Lesley J. Yellowlees, Simon Daff, and Stephen K. Chapman

From the Department of Chemistry, University of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, Scotland, United Kingdom

The nitrogenous -acceptor ligand 4-cyanopyridine (4CNPy) exhibits reversible ligation to ferrous heme in the flavocytochrome P450 BM3 (K_d = 1.8 µM 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 µM (cf. K_d(ox) = 364 µM). 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.

Received for publication, July 29, 2004 , and in revised form, September 7, 2004.

^* This work was supported by the UK Biotechnology and Biological Sciences Research Council and by the Edinburgh Protein Interaction Centre. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 44-131-650-4760; Fax: 44-131-650-6453; E-mail: s.k.chapman{at}ed.ac.uk.

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