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. 2000 Jan 15;345(Pt 2):393–399.

Initial-rate kinetics of the flavin reductase reaction catalysed by human biliverdin-IXbeta reductase (BVR-B).

O Cunningham 1, M G Gore 1, T J Mantle 1
PMCID: PMC1220769  PMID: 10620517

Abstract

The initial-rate kinetics of the flavin reductase reaction catalysed by biliverdin-IXbeta reductase at pH 7.5 are consistent with a rapid-equilibrium ordered mechanism, with the pyridine nucleotide binding first. NADPH binding to the free enzyme was characterized using stopped-flow fluorescence quenching, and a K(d) of 15.8 microM was calculated. Equilibrium fluorescence quenching experiments indicated a K(d) of 0.55 microM, suggesting that an enzyme-NADPH encounter complex (K(d) 15.8 microM) isomerizes to a more stable 'nucleotide-induced' conformation. The enzyme was shown to catalyse the reduction of FMN, FAD and riboflavin, with K(m) values of 52 microM, 125 microM and 53 microM, respectively. Lumichrome was shown to be a competitive inhibitor against FMN, with a K(i) of 76 microM, indicating that interactions with the isoalloxazine ring are probably sufficient for binding. During initial experiments it was observed that both the flavin reductase and biliverdin reductase activities of the enzyme exhibit a sharp optimum at pH 5 in citrate buffer. An initial-rate study indicated that the enzyme obeys a steady-state ordered mechanism in this buffer. The initial-rate kinetics in sodium acetate at pH 5 are consistent with a rapid-equilibrium ordered mechanism, indicating that citrate may directly affect the enzyme's behaviour at pH 5. Mesobiliverdin XIIIalpha, a synthetic biliverdin which binds to flavin reductase but does not act as a substrate for the enzyme, exhibits competitive kinetics with FMN (K(i) 0.59 microM) and mixed-inhibition kinetics with NADPH. This is consistent with a single pyridine nucleotide site and competition by FMN and biliverdin for a second site. Interestingly, flavin reductase/biliverdin-IXbeta reductase has also been shown to exhibit ferric reductase activity, with an apparent K(m) of 2.5 microM for the ferric iron. The ferric reductase reaction requires NAD(P)H and FMN. This activity is intriguing, as haem cleavage in the foetus produces non-alpha isomers of biliverdin and ferric iron, both of which are substrates for flavin reductase/biliverdin-IXbeta reductase.

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Selected References

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  1. Blanckaert N., Heirwegh K. P., Zaman Z. Comparison of the biliary excretion of the four isomers of bilirubin-IX in Wistar and homozygous Gunn rats. Biochem J. 1977 Apr 15;164(1):229–236. doi: 10.1042/bj1640229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brodersen R., Bartels P. Enzymatic oxidation of bilirubin. Eur J Biochem. 1969 Oct;10(3):468–473. doi: 10.1111/j.1432-1033.1969.tb00712.x. [DOI] [PubMed] [Google Scholar]
  3. Coves J., Fontecave M. Reduction and mobilization of iron by a NAD(P)H:flavin oxidoreductase from Escherichia coli. Eur J Biochem. 1993 Feb 1;211(3):635–641. doi: 10.1111/j.1432-1033.1993.tb17591.x. [DOI] [PubMed] [Google Scholar]
  4. Cunningham O., Mantle T. J. Cloning, overexpression and purification of biliverdin IX-beta reductase. Biochem Soc Trans. 1997 Nov;25(4):S613–S613. doi: 10.1042/bst025s613. [DOI] [PubMed] [Google Scholar]
  5. DeFilippi L. J., Hultquist D. E. The green hemoproteins of bovine erythrocytes. I purification and characterization. J Biol Chem. 1978 May 10;253(9):2946–2953. [PubMed] [Google Scholar]
  6. DeFilippi L. J., Hultquist D. E. The green hemoproteins of bovine erythrocytes. II. Spectral, ligand-binding, and electrochemical properties. J Biol Chem. 1978 May 10;253(9):2954–2962. [PubMed] [Google Scholar]
  7. FLORINI J. R., VESTLING C. S. Graphical determination of the dissociation constants for two-substrate enzyme systems. Biochim Biophys Acta. 1957 Sep;25(3):575–578. doi: 10.1016/0006-3002(57)90529-2. [DOI] [PubMed] [Google Scholar]
  8. Fontecave M., Eliasson R., Reichard P. NAD(P)H:flavin oxidoreductase of Escherichia coli. A ferric iron reductase participating in the generation of the free radical of ribonucleotide reductase. J Biol Chem. 1987 Sep 5;262(25):12325–12331. [PubMed] [Google Scholar]
  9. Hunt S. C., Wu L. L., Hopkins P. N., Williams R. R. Evidence for a major gene elevating serum bilirubin concentration in Utah pedigrees. Arterioscler Thromb Vasc Biol. 1996 Aug;16(8):912–917. doi: 10.1161/01.atv.16.8.912. [DOI] [PubMed] [Google Scholar]
  10. Jacobsen J., Fedders O. Determination of non-albumin-bound bilirubin in human serum. Scand J Clin Lab Invest. 1970 Nov;26(3):237–241. doi: 10.3109/00365517009046228. [DOI] [PubMed] [Google Scholar]
  11. Levine R. L. Fluorescence-quenching studies of the binding of bilirubin to albumin. Clin Chem. 1977 Dec;23(12):2292–2301. [PubMed] [Google Scholar]
  12. Mack C. P., Hultquist D. E., Shlafer M. Myocardial flavin reductase and riboflavin: a potential role in decreasing reoxygenation injury. Biochem Biophys Res Commun. 1995 Jul 6;212(1):35–40. doi: 10.1006/bbrc.1995.1932. [DOI] [PubMed] [Google Scholar]
  13. Neuzil J., Stocker R. Free and albumin-bound bilirubin are efficient co-antioxidants for alpha-tocopherol, inhibiting plasma and low density lipoprotein lipid peroxidation. J Biol Chem. 1994 Jun 17;269(24):16712–16719. [PubMed] [Google Scholar]
  14. Schwertner H. A., Jackson W. G., Tolan G. Association of low serum concentration of bilirubin with increased risk of coronary artery disease. Clin Chem. 1994 Jan;40(1):18–23. [PubMed] [Google Scholar]
  15. Shalloe F., Elliott G., Ennis O., Mantle T. J. Evidence that biliverdin-IX beta reductase and flavin reductase are identical. Biochem J. 1996 Jun 1;316(Pt 2):385–387. doi: 10.1042/bj3160385. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Stevens B., Small R. D., Jr The photoperoxidation of unsaturated organic molecules--XV. O21Delta g quenching by bilirubin and biliverdin. Photochem Photobiol. 1976 Jan;23(1):33–36. doi: 10.1111/j.1751-1097.1976.tb06767.x. [DOI] [PubMed] [Google Scholar]
  17. Stocker R., Glazer A. N., Ames B. N. Antioxidant activity of albumin-bound bilirubin. Proc Natl Acad Sci U S A. 1987 Aug;84(16):5918–5922. doi: 10.1073/pnas.84.16.5918. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Yamaguchi T., Komoda Y., Nakajima H. Biliverdin-IX alpha reductase and biliverdin-IX beta reductase from human liver. Purification and characterization. J Biol Chem. 1994 Sep 30;269(39):24343–24348. [PubMed] [Google Scholar]
  19. Yamaguchi T., Komuro A., Nakano Y., Tomita M., Nakajima H. Complete amino acid sequence of biliverdin-IX beta reductase from human liver. Biochem Biophys Res Commun. 1993 Dec 30;197(3):1518–1523. doi: 10.1006/bbrc.1993.2649. [DOI] [PubMed] [Google Scholar]
  20. Yamaguchi T., Nakajima H. Changes in the composition of bilirubin-IX isomers during human prenatal development. Eur J Biochem. 1995 Oct 15;233(2):467–472. doi: 10.1111/j.1432-1033.1995.467_2.x. [DOI] [PubMed] [Google Scholar]

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