Biophysical characterization of the sterol demethylase P450 from Mycobacterium tuberculosis, its cognate ferredoxin, and their interactions.
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 epsilon419 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 (Kd = 21.7 microM). 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 approximately 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.
McLean
Kirsty J
KJ
Manchester Interdisciplinary Biocentre, School of Chemical Engineering and Analytical Sciences, University of Manchester, Jackson's Mill, P.O. Box 88, Sackville Street, Manchester M60 1QD, UK.
Warman
Ashley J
AJ
Seward
Harriet E
HE
Marshall
Ker R
KR
Girvan
Hazel M
HM
Cheesman
Myles R
MR
Waterman
Michael R
MR
Munro
Andrew W
AW
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D002248:Carbon Monoxide / Q000737:chemistry; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics; D005288:Ferredoxins / Q000737:chemistry*; D006418:Heme / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000737:chemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics; D011199:Potentiometry; D013057:Spectrum Analysis; D058886:Sterol 14-Demethylase
D001426:Bacterial Proteins; D005288:Ferredoxins; D006418:Heme; D002248:Carbon Monoxide; D003577:Cytochrome P-450 Enzyme System; D007501:Iron; D010088:Oxidoreductases; D058886:Sterol 14-Demethylase