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Equilibrium and transient state spectrophotometric studies of the mechanism of reduction of the flavoprotein domain of P450BM-3.
Biochemistry. 1996 Jun 04; 35(22):7058-68.B

Abstract

The flavoprotein domain of P450BM-3 (BMR), which is functionally analogous to eukaryotic NADPH-P450 oxidoreductases, contains both FAD and FMN. When BMR is titrated with NADPH or sodium dithionite under anaerobic conditions, addition of 2 electron equivalents per mole of BMR results in the reduction of the high potential flavin (FMN) without the accumulation of semiquinone intermediates. Additional sodium dithionite first produces some neutral, blue flavin semiquinone radical and, finally, fully reduced FADH2. During reduction with NADPH, an absorbance increase characteristic of the formation of a flavin-pyridine nucleotide charge-transfer complex was observed only during the addition of the second mole of NADPH per mole of BMR. On the basis of these results, we conclude that the midpoint reduction potential for the FMN semiquinone/FMNH2 couple is more positive than that for FMN/FMN semiquinone. The kinetics of reduction of BMR with NADPH were studied by stopped-flow spectrophotometry. With a 1:1 ratio of NADPH to BMR, the absorbance changes can be fit to five consecutive first order reactions with rate constants of 350 s-1, 130 s-1, 27 s-1, 2.3 s-1, and 0.05 s-1. These reactions are most probably the following: (a) complex formation between BMR and NADPH; (b) reduction of FAD with formation of the NADP(+)-FADH- charge-transfer complex; (c) transfer of the first electron from FADH- to FMN to form an anionic, red FMN semiquinone leaving the FAD as the neutral, blue semiquinone. Precise identification of intermediates beyond this point is difficult. In the presence of a 10-fold molar excess of NADPH, the absorbance changes and rate constants are somewhat different due to the formation of several additional reduced species of BMR. The rate of the first step increases, confirming that this is the formation of the NADPH-BMR complex. Our results indicate that the kinetic and thermodynamic control of the flavins in BMR is significantly different from that in microsomal P450 reductase. The low potential of the anionic FMN semiquinone can be utilized to reduce the P450 heme. When the anionic semiquinone becomes protonated, its potential becomes more positive and it is readily reduced to FMNH2, which is not capable of reducing P450.

Authors+Show Affiliations

Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas 75235-9038, USA.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

8679531

Citation

Sevrioukova, I, et al. "Equilibrium and Transient State Spectrophotometric Studies of the Mechanism of Reduction of the Flavoprotein Domain of P450BM-3." Biochemistry, vol. 35, no. 22, 1996, pp. 7058-68.
Sevrioukova I, Shaffer C, Ballou DP, et al. Equilibrium and transient state spectrophotometric studies of the mechanism of reduction of the flavoprotein domain of P450BM-3. Biochemistry. 1996;35(22):7058-68.
Sevrioukova, I., Shaffer, C., Ballou, D. P., & Peterson, J. A. (1996). Equilibrium and transient state spectrophotometric studies of the mechanism of reduction of the flavoprotein domain of P450BM-3. Biochemistry, 35(22), 7058-68.
Sevrioukova I, et al. Equilibrium and Transient State Spectrophotometric Studies of the Mechanism of Reduction of the Flavoprotein Domain of P450BM-3. Biochemistry. 1996 Jun 4;35(22):7058-68. PubMed PMID: 8679531.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Equilibrium and transient state spectrophotometric studies of the mechanism of reduction of the flavoprotein domain of P450BM-3. AU - Sevrioukova,I, AU - Shaffer,C, AU - Ballou,D P, AU - Peterson,J A, PY - 1996/6/4/pubmed PY - 1996/6/4/medline PY - 1996/6/4/entrez SP - 7058 EP - 68 JF - Biochemistry JO - Biochemistry VL - 35 IS - 22 N2 - The flavoprotein domain of P450BM-3 (BMR), which is functionally analogous to eukaryotic NADPH-P450 oxidoreductases, contains both FAD and FMN. When BMR is titrated with NADPH or sodium dithionite under anaerobic conditions, addition of 2 electron equivalents per mole of BMR results in the reduction of the high potential flavin (FMN) without the accumulation of semiquinone intermediates. Additional sodium dithionite first produces some neutral, blue flavin semiquinone radical and, finally, fully reduced FADH2. During reduction with NADPH, an absorbance increase characteristic of the formation of a flavin-pyridine nucleotide charge-transfer complex was observed only during the addition of the second mole of NADPH per mole of BMR. On the basis of these results, we conclude that the midpoint reduction potential for the FMN semiquinone/FMNH2 couple is more positive than that for FMN/FMN semiquinone. The kinetics of reduction of BMR with NADPH were studied by stopped-flow spectrophotometry. With a 1:1 ratio of NADPH to BMR, the absorbance changes can be fit to five consecutive first order reactions with rate constants of 350 s-1, 130 s-1, 27 s-1, 2.3 s-1, and 0.05 s-1. These reactions are most probably the following: (a) complex formation between BMR and NADPH; (b) reduction of FAD with formation of the NADP(+)-FADH- charge-transfer complex; (c) transfer of the first electron from FADH- to FMN to form an anionic, red FMN semiquinone leaving the FAD as the neutral, blue semiquinone. Precise identification of intermediates beyond this point is difficult. In the presence of a 10-fold molar excess of NADPH, the absorbance changes and rate constants are somewhat different due to the formation of several additional reduced species of BMR. The rate of the first step increases, confirming that this is the formation of the NADPH-BMR complex. Our results indicate that the kinetic and thermodynamic control of the flavins in BMR is significantly different from that in microsomal P450 reductase. The low potential of the anionic FMN semiquinone can be utilized to reduce the P450 heme. When the anionic semiquinone becomes protonated, its potential becomes more positive and it is readily reduced to FMNH2, which is not capable of reducing P450. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/8679531/Equilibrium_and_transient_state_spectrophotometric_studies_of_the_mechanism_of_reduction_of_the_flavoprotein_domain_of_P450BM_3_ L2 - https://doi.org/10.1021/bi960060a DB - PRIME DP - Unbound Medicine ER -