Biophysical Characterization of the Sterol Demethylase P450 from Mycobacterium tuberculosis, Its Cognate Ferredoxin, and Their Interactions

• Published in: Biochemistry (Easton) Vol. 45; no. 27; pp. 8427 - 8443
• Main Authors: McLean, Kirsty J, Warman, Ashley J, Seward, Harriet E, Marshall, Ker R, Girvan, Hazel M, Cheesman, Myles R, Waterman, Michael R, Munro, Andrew W
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 11.07.2006
• Subjects: Amino Acid Sequence; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Carbon Monoxide - chemistry; Cytochrome P-450 Enzyme System - chemistry; Cytochrome P-450 Enzyme System - genetics; Ferredoxins - chemistry; Heme - chemistry; Hydrogen-Ion Concentration; Iron - chemistry; Kinetics; Molecular Sequence Data; Mycobacterium tuberculosis; Mycobacterium tuberculosis - enzymology; Oxidation-Reduction; Oxidoreductases - chemistry; Oxidoreductases - genetics; Potentiometry; Spectrum Analysis; Sterol 14-Demethylase
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi0601609

Mycobacterium tuberculosis encodes a P450 of the sterol demethylase family (CYP51) chromosomally located adjacent to a ferredoxin (Fdx). CYP51 and Fdx were purified to homogeneity and characterized. Spectroscopic analyses were consistent with cysteinate- and aqua-ligated heme iron in CYP51. An ε419 of 134 mM-1 cm-1 was determined for oxidized CYP51. Analysis of interactions of 1-, 2-, and 4-phenylimidazoles with CYP51 showed that the 1- and 4-forms were heme iron-coordinating inhibitors, while 2-phenylimidazole induced a substrate-like optical shift. The 2-phenyimidazole-bound CYP51 demonstrated unusual decreases in high-spin heme iron content at elevated temperatures and an almost complete absence of high-spin heme iron by low-temperature EPR. These data suggest thermally induced alterations in CYP51 active site structure and/or binding modes for the small ligand. Reduction of CYP51 in the presence of carbon monoxide leads to formation of an Fe(II)−CO complex with a Soret absorption maximum at 448.5 nm, which collapses (at 0.246 min-1 at pH 7.0) forming a species with a Soret maximum at 421.5 nm (the inactive P420 form). The rate of P420 formation is accelerated at lower pH, consistent with protonation of the cysteinate (Cys 394) to a thiol underlying the P450−P420 transition. The P450 form is stabilized by estriol, which induces a type I spectral shift on binding CYP51 (K d = 21.7 μM). Nonstandard spectral changes occur on CYP51 reduction (using either dithionite or natural redox partners), including a blue-shifted Soret band and development of a strong feature at ∼558.5 nm, suggestive of cysteine thiol ligation. Thus, ligand-free ferrous CYP51 is prone to thiolate ligand protonation even in the absence of carbon monoxide. Analysis of reoxidized CYP51 demonstrates that the enzyme re-forms P450, indicating that Cys 394 thiol is readily deprotonated to thiolate in the ferric form. Spectroscopic analysis of Fdx by EPR (resonance at g = 2.03) and magnetic CD (intensity for oxidized and reduced forms and signal intensity dependence on field strength and temperature) demonstrated that Fdx binds a [3Fe-4S] iron−sulfur cluster. Potentiometric studies show that the midpoint potential for ligand-free CYP51 is −375 mV, increasing to −225 mV in the estriol-bound form. The Fdx potential is −31 mV. Fdx forms a productive electron transfer complex with CYP51 and reduces it at a rate of 3.0 min-1 in the ligand-free form and 4.3 min-1 in the estriol-bound form, despite a thermodynamic barrier. Steady-state analysis of a M. tuberculosis class I redox system comprising flavoprotein reductase A (FprA), Fdx, and estriol-bound CYP51 indicates heme iron reduction as a rate-limiting step.