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Kinetic, spectroscopic and thermodynamic characterization of the Mycobacterium tuberculosis adrenodoxin reductase homologue FprA.
Biochem J. 2003 Jun 01; 372(Pt 2):317-27.BJ

Abstract

The genome sequence of the pathogenic bacterium Mycobacterium tuberculosis revealed numerous cytochrome P450 enzymes, which require accessory redox enzymes for catalytic function (ferredoxin reductase and ferredoxin). The most likely ferredoxin reductase is encoded by fprA, and its structure resembles eukaryotic adrenodoxin reductases. We have cloned, expressed and purified the flavoenzyme product of the fprA gene in Escherichia coli. FprA reduces various electron acceptors using either NADPH or NADH as the electron donor, but discriminates in favour of NADPH (apparent K (m) for NADH=50.6+/-3.1 microM; NADPH=4.1+/-0.3 microM from ferricyanide reduction experiments). Stopped-flow studies of reduction of the FprA FAD by NADPH demonstrate increased flavin reduction rate at low NADPH concentration (<200 microM), consistent with the presence of a second, kinetically distinct and inhibitory, pyridine nucleotide-binding site, similar to that identified in human cytochrome P450 reductase [Gutierrez, Lian, Wolf, Scrutton and Roberts (2001) Biochemistry 40, 1964-1975]. Flavin reduction by NADH is slower than with NADPH and displays hyperbolic dependence on NADH concentration [maximal reduction rate (k (red))=25.4+/-0.7 s(-1), apparent K (d)=42.9+/-4.6 microM]. Flavin reoxidation by molecular oxygen is more rapid for NADH-reduced enzyme. Reductive titrations show that the enzyme forms a species with spectral characteristics typical of a neutral (blue) FAD semiquinone only on reduction with NADPH, consistent with EPR studies. The second order dependence of semiquinone formation on the concentration of FprA indicates a disproportionation reaction involving oxidized and two-electron-reduced FprA. Titration of FprA with dithionite converts oxidized FAD into the hydroquinone form; the flavin semiquinone is not populated under these conditions. The midpoint reduction potential for the two electron couple is -235+/-5 mV (versus the normal hydrogen electrode), similar to that for adrenodoxin reductase (-274 mV). Our data provide a thermodynamic and transient kinetic framework for catalysis by FprA, and complement recent spectrophotometric and steady-state studies of the enzyme [Fischer, Raimondi, Aliverti and Zanetti (2002) Eur. J. Biochem. 269, 3005-3013].

Authors+Show Affiliations

Department of Biochemistry, University of Leicester, The Adrian Building, University Road, UK.No affiliation info availableNo affiliation info available

Pub Type(s)

Comparative Study
Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

12614197

Citation

McLean, Kirsty J., et al. "Kinetic, Spectroscopic and Thermodynamic Characterization of the Mycobacterium Tuberculosis Adrenodoxin Reductase Homologue FprA." The Biochemical Journal, vol. 372, no. Pt 2, 2003, pp. 317-27.
McLean KJ, Scrutton NS, Munro AW. Kinetic, spectroscopic and thermodynamic characterization of the Mycobacterium tuberculosis adrenodoxin reductase homologue FprA. Biochem J. 2003;372(Pt 2):317-27.
McLean, K. J., Scrutton, N. S., & Munro, A. W. (2003). Kinetic, spectroscopic and thermodynamic characterization of the Mycobacterium tuberculosis adrenodoxin reductase homologue FprA. The Biochemical Journal, 372(Pt 2), 317-27.
McLean KJ, Scrutton NS, Munro AW. Kinetic, Spectroscopic and Thermodynamic Characterization of the Mycobacterium Tuberculosis Adrenodoxin Reductase Homologue FprA. Biochem J. 2003 Jun 1;372(Pt 2):317-27. PubMed PMID: 12614197.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Kinetic, spectroscopic and thermodynamic characterization of the Mycobacterium tuberculosis adrenodoxin reductase homologue FprA. AU - McLean,Kirsty J, AU - Scrutton,Nigel S, AU - Munro,Andrew W, PY - 2003/03/03/accepted PY - 2003/02/28/revised PY - 2002/10/30/received PY - 2003/3/5/pubmed PY - 2003/7/23/medline PY - 2003/3/5/entrez SP - 317 EP - 27 JF - The Biochemical journal JO - Biochem J VL - 372 IS - Pt 2 N2 - The genome sequence of the pathogenic bacterium Mycobacterium tuberculosis revealed numerous cytochrome P450 enzymes, which require accessory redox enzymes for catalytic function (ferredoxin reductase and ferredoxin). The most likely ferredoxin reductase is encoded by fprA, and its structure resembles eukaryotic adrenodoxin reductases. We have cloned, expressed and purified the flavoenzyme product of the fprA gene in Escherichia coli. FprA reduces various electron acceptors using either NADPH or NADH as the electron donor, but discriminates in favour of NADPH (apparent K (m) for NADH=50.6+/-3.1 microM; NADPH=4.1+/-0.3 microM from ferricyanide reduction experiments). Stopped-flow studies of reduction of the FprA FAD by NADPH demonstrate increased flavin reduction rate at low NADPH concentration (<200 microM), consistent with the presence of a second, kinetically distinct and inhibitory, pyridine nucleotide-binding site, similar to that identified in human cytochrome P450 reductase [Gutierrez, Lian, Wolf, Scrutton and Roberts (2001) Biochemistry 40, 1964-1975]. Flavin reduction by NADH is slower than with NADPH and displays hyperbolic dependence on NADH concentration [maximal reduction rate (k (red))=25.4+/-0.7 s(-1), apparent K (d)=42.9+/-4.6 microM]. Flavin reoxidation by molecular oxygen is more rapid for NADH-reduced enzyme. Reductive titrations show that the enzyme forms a species with spectral characteristics typical of a neutral (blue) FAD semiquinone only on reduction with NADPH, consistent with EPR studies. The second order dependence of semiquinone formation on the concentration of FprA indicates a disproportionation reaction involving oxidized and two-electron-reduced FprA. Titration of FprA with dithionite converts oxidized FAD into the hydroquinone form; the flavin semiquinone is not populated under these conditions. The midpoint reduction potential for the two electron couple is -235+/-5 mV (versus the normal hydrogen electrode), similar to that for adrenodoxin reductase (-274 mV). Our data provide a thermodynamic and transient kinetic framework for catalysis by FprA, and complement recent spectrophotometric and steady-state studies of the enzyme [Fischer, Raimondi, Aliverti and Zanetti (2002) Eur. J. Biochem. 269, 3005-3013]. SN - 0264-6021 UR - https://www.unboundmedicine.com/medline/citation/12614197/Kinetic_spectroscopic_and_thermodynamic_characterization_of_the_Mycobacterium_tuberculosis_adrenodoxin_reductase_homologue_FprA_ L2 - https://portlandpress.com/biochemj/article-lookup/doi/10.1042/BJ20021692 DB - PRIME DP - Unbound Medicine ER -