Rapid P450 Heme Iron Reduction by Laser Photoexcitation of Mycobacterium tuberculosis CYP121 and CYP51B1: Analysis of CO complexation reactions and reversibility of the P450/P420 equilibrium

Adrian J. Dunford, Kirsty J. McLean, Muna Sabri, Harriet E. Seward, Derren J. Heyes, Nigel S. Scrutton, Andrew W. Munro

Research output: Contribution to journalArticle

39 Citations (Scopus)

Abstract

We demonstrate that photoexcitation of NAD(P)H reduces heme iron of Mycobacterium tuberculosis P450s CYP121 and CYP51B1 on the microsecond time scale. Rates of formation for the ferrous-carbonmonoxy (FeII-CO) complex were determined across a range of coenzyme/CO concentrations. CYP121 reaction transients were biphasic. A hyperbolic dependence on CO concentration was observed, consistent with the presence of a CO binding site in ferric CYP121. CYP51B1 absorption transients for FeII-CO complex formation were monophasic. The reaction rate was second order with respect to [CO], suggesting the absence of a CO-binding site in ferric CYP51B1. In the absence of CO, heme iron reduction by photoexcited NAD(P)H is fast (∼10,000-11,000 s-1) with both P450s. For CYP121, transients revealed initial production of the thiolate-coordinated (P450) complex (absorbance maximum at 448 nm), followed by a slower phase reporting partial conversion to the thiol-coordinated P420 species (at 420 nm). The slow phase amplitude increased at lower pH values, consistent with heme cysteinate protonation underlying the transition. Thus, CO binding occurs to the thiolate-coordinated ferrous form prior to cysteinate protonation. For CYP51B1, slow conversions of both the ferrous/FeII-CO forms to species with spectral maxima at 423/421.5 nm occurred following photoexcitation in the absence/presence of CO. This reflected conversion from ferrous thiolate- to thiol-coordinated forms in both cases, indicating instability of the thiolate-coordinated ferrous CYP51B1. CYP121 FeII-CO complex pH titrations revealed reversible spectral transitions between P450 and P420 forms. Our data provide strong evidence for P420 formation linked to reversible heme thiolate protonation, and demonstrate key differences in heme chemistry and CO binding for CYP121 and CYP51B1.

Original languageEnglish
Pages (from-to)24816-24824
Number of pages9
JournalJournal of Biological Chemistry
Volume282
Issue number34
Early online date6 Jun 2007
DOIs
Publication statusPublished - 24 Aug 2007
Externally publishedYes

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