Efficient catalytic turnover of cytochrome P450cam is supported by a T252N mutation

Abstract

A Thr (or Ser) residue on the I-helix is a highly conserved structural feature of cytochrome P450 enzymes. It is believed to be indispensable as a proton delivery shuttle in the oxygen activation process. Previous work showed that P450 cin (CYP176A1), which contains an Asn instead of the conserved Thr, is fully functional in the catalytic oxidation of cineole [Hawkes, D.B. et al. (2002) J. Biol. Chem. 277, 27725-27732]. To determine whether the substitution of Asn for Thr is specific or general, the conserved Thr252 in P450 cam (CYP101) was mutated to generate the T252N, T252N/ V253T, and T252A mutants. Steady-state kinetic analysis of the oxidation of camphor by these mutants indicated that the T252N and T252N/V253T mutants have comparable turnover numbers but higher K m values relative to the wild-type enzyme. Spectroscopic binding assays indicate that the higher K m values reflect a decrease in the camphor binding affinity. Non-productive H 2 O 2 generation was negligible with the T252N and T252N/V253T mutants, but, as previously observed, was dominant in the T252A mutant. Our results, and a structure model based on the crystal structures of the ferrous dioxygen complexes of P450 cam and its T252A mutant, suggest that Asn252 can stabilize the ferric hydroperoxy intermediate, preventing premature release of H 2 O 2 and enabling addition of the second proton to the distal oxygen to generate the catalytic ferryl species.The cytochrome P450 (CYP or P450) family of enzymes is involved in oxidative metabolism of a wide range of endo-and exogenous chemicals [1]. P450 cam (CYP101) from Pseudomonas putida, the structurally and biochemically best characterized P450 enzyme [2], catalyzes the regio-and stereospecific hydroxylation of camphor to 5-exo-hydroxycamphor. As first established for P450 cam , the general P450 catalytic cycle involves (a) substrate binding, (b) reduction of the iron from the ferric to the ferrous state, (c) Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.Abbreviations used: CYP (P450), cytochrome P450; PCR, polymerase chain reaction; ORF, open reading frames; Pd, putidaredoxin; PdR, putidaredoxin reductase; TB, Terrific Broth; GC-MS, gas chromatography-mass spectrometry. K d indicates a dissociation constant estimated by measurement of the UV-visible changes in the P450 heme spectrum. An acid-alcohol side-chain pair, typically an Asp and Thr, is thought in most P450 enzymes to be a requirement for productive dioxygen activation in P450 catalysis [3]. The crystal structures of ferrous camphor-and CO-bound P450 cam suggest that Thr252 is a key catal...

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