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 (Fe(II)-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 Fe(II)-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 ( approximately 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/Fe(II)-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 Fe(II)-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.
Highlights
We demonstrate that photoexcitation of NAD(P)H reduces heme iron of Mycobacterium tuberculosis P450s CYP121 and CYP51B1 on the microsecond time scale
We have exploited this method to analyze the kinetics of P450 reduction, FeII-CO complex formation, and the P450 to P420 collapse in the Mycobacterium tuberculosis (Mtb) CYP51B1 and CYP121 P450s
In view of protonation of cysteine thiolate likely underlying the formation of P420 in CYP121, we examined the decay of P450 to P420 across a range of pH values and with single wavelength analysis at 450 nm
Summary
Recent studies of the Sorangium cellulosum P450epoK (CYP167A1) indicate that the time-dependent conversion of the P450 FeII-CO complex to the P420 form can be reversed in the presence of the substrate epothilone D, leading to “substrate-mediated rescue” of the inactivated protein and formation of substantial amounts of the P450 form with an absorbance maximum at 446 nm [20]. We demonstrate, for both enzymes, that the P450 FeII-CO complex is formed rapidly on laser photoexcitation of the oxidized enzyme in the presence of CO This is followed by a slower process of conversion to P420, demonstrating that the heme thiolate protonation is a consequence of heme iron reduction and/or CO coordination, and that there is no significant amount of thiol coordination of ferric heme iron in the resting forms of either P450. We demonstrate that the CYP121 P450/P420 equilibrium is reversible and exquisitely pH-dependent, reinforcing the importance of thiolate protonation in the formation of the cytochrome P420 species
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