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J. Biol. Chem., Vol. 280, Issue 51, 42134-42141, December 23, 2005

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Direct Observation of a Novel Perturbed Oxyferrous Catalytic Intermediate during Reduced Putidaredoxin-initiated Turnover of Cytochrome P-450-CAM

PROBING THE EFFECTOR ROLE OF PUTIDAREDOXIN IN CATALYSIS^*

Mary C. Glascock, David P. Ballou1, and John H. Dawson¶2

From the Department of Chemistry and Biochemistry and the ^¶School of Medicine, University of South Carolina, Columbia, South Carolina 29208 and the Department of Biological Chemistry, Medical School, University of Michigan, Ann Arbor, Michigan 48109-0606

The single turnover of (1R)(+)-camphor-bound oxyferrous cytochrome P450-CAM with one equivalent of dithionite-reduced putidaredoxin (Pdx) was monitored for the appearance of transient intermediates at 3 °C by double mixing rapid scanning stopped-flow spectroscopy. With excess camphor, three successive species were observed after generating oxyferrous P450-CAM and reacting versus reduced Pdx: a perturbed oxyferrous derivative, a species that was a mixture of high and low spin Fe(III), and high spin ferric camphor-bound enzyme. The rates of the first two steps, 140 and 85 s^-1, were assigned to formation of the perturbed oxyferrous intermediate and to electron transfer from reduced Pdx, respectively. In the presence of stoichiometric substrate, three phases with similar rates were seen even though the final state is low spin ferric P450-CAM. This is consistent with substrate being hydroxylated during the reaction. The single turnover reaction initiated by adding dioxygen to a preformed reduced P450-CAM·Pdx complex with excess camphor also led to phases with similar rates. It is proposed that formation of the perturbed oxyferrous intermediate reflects alteration of H-bonding to the proximal Cys, increasing the reduction potential of the oxyferrous state and triggering electron transfer from reduced Pdx. This species may be a direct spectral signature of the effector role of Pdx on P450-CAM reactivity (i.e. during catalysis). The substrate-free oxyferrous enzyme also reacted readily with reduced Pdx, showing that the inability of substrate-free P450-CAM to accept electrons from reduced Pdx and function as an NADH oxidase is completely due to the incapacity of reduced Pdx to deliver the first but not the second electron.

Received for publication, May 17, 2005 , and in revised form, August 19, 2005.

^* This work was supported by National Institutes of Health Grants GM 20877 (to D. P. B.) and GM 26730 (to J. H. D.). 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.

¹ To whom correspondence may be addressed. Tel.: 734-764-9582; Fax: 734-763-4581; E-mail: dballou{at}umich.edu. ² To whom correspondence may be addressed. Tel.: 803-777-7234; Fax: 803-777-9521; E-mail: dawson{at}sc.edu.

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