Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 2002 Oct 1;367(Pt 1):19–30. doi: 10.1042/BJ20020667

Stopped-flow kinetic studies of electron transfer in the reductase domain of neuronal nitric oxide synthase: re-evaluation of the kinetic mechanism reveals new enzyme intermediates and variation with cytochrome P450 reductase.

Kirsty Knight 1, Nigel S Scrutton 1
PMCID: PMC1222855  PMID: 12079493

Abstract

The reduction by NADPH of the FAD and FMN redox centres in the isolated flavin reductase domain of calmodulin-bound rat neuronal nitric oxide synthase (nNOS) has been studied by anaerobic stopped-flow spectroscopy using absorption and fluorescence detection. We show by global analysis of time-dependent photodiode array spectra, single wavelength absorption and NADPH fluorescence studies, that at least four resolvable steps are observed in stopped-flow studies with NADPH and that flavin reduction is reversible. The first reductive step represents the rapid formation of an equilibrium between an NADPH-enzyme charge-transfer species and two-electron-reduced enzyme bound to NADP(+). The second and third steps represent further reduction of the enzyme flavins and NADP(+) release. The fourth step is attributed to the slow accumulation of an enzyme species that is inferred not to be relevant catalytically in steady-state reactions. Stopped-flow flavin fluorescence studies indicate the presence of slow kinetic phases, the timescales of which correspond to the slow phase observed in absorption and NADPH fluorescence transients. By analogy with stopped-flow studies of cytochrome P450 reductase, we attribute these slow fluorescence and absorption changes to enzyme disproportionation and/or conformational change. Unlike for the functionally related cytochrome P450 reductase, transfer of the first hydride equivalent from NADPH to nNOS reductase does not generate the flavin di-semiquinoid state. This indicates that internal electron transfer is relatively slow and is probably gated by NADP(+) release. Release of calmodulin from the nNOS reductase does not affect the kinetics of inter-flavin electron transfer under stopped-flow conditions, although the observed rate of formation of the equilibrium between the NADPH-oxidized enzyme charge-transfer species and two-electron-reduced enzyme bound to NADP(+) is modestly slower in calmodulin-depleted enzyme. Our studies indicate the need for significant re-interpretation of published kinetic data for electron transfer in the reductase domain of neuronal nitric oxide synthase.

Full Text

The Full Text of this article is available as a PDF (355.6 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abu-Soud H. M., Stuehr D. J. Nitric oxide synthases reveal a role for calmodulin in controlling electron transfer. Proc Natl Acad Sci U S A. 1993 Nov 15;90(22):10769–10772. doi: 10.1073/pnas.90.22.10769. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Abu-Soud H. M., Yoho L. L., Stuehr D. J. Calmodulin controls neuronal nitric-oxide synthase by a dual mechanism. Activation of intra- and interdomain electron transfer. J Biol Chem. 1994 Dec 23;269(51):32047–32050. [PubMed] [Google Scholar]
  3. Barna T. M., Khan H., Bruce N. C., Barsukov I., Scrutton N. S., Moody P. C. Crystal structure of pentaerythritol tetranitrate reductase: "flipped" binding geometries for steroid substrates in different redox states of the enzyme. J Mol Biol. 2001 Jul 6;310(2):433–447. doi: 10.1006/jmbi.2001.4779. [DOI] [PubMed] [Google Scholar]
  4. Bredt D. S., Hwang P. M., Glatt C. E., Lowenstein C., Reed R. R., Snyder S. H. Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase. Nature. 1991 Jun 27;351(6329):714–718. doi: 10.1038/351714a0. [DOI] [PubMed] [Google Scholar]
  5. Bredt D. S., Snyder S. H. Nitric oxide: a physiologic messenger molecule. Annu Rev Biochem. 1994;63:175–195. doi: 10.1146/annurev.bi.63.070194.001135. [DOI] [PubMed] [Google Scholar]
  6. Brunner K., Tortschanoff A., Hemmens B., Andrew P. J., Mayer B., Kungl A. J. Sensitivity of flavin fluorescence dynamics in neuronal nitric oxide synthase to cofactor-induced conformational changes and dimerization. Biochemistry. 1998 Dec 15;37(50):17545–17553. doi: 10.1021/bi981138l. [DOI] [PubMed] [Google Scholar]
  7. Cho H. J., Xie Q. W., Calaycay J., Mumford R. A., Swiderek K. M., Lee T. D., Nathan C. Calmodulin is a subunit of nitric oxide synthase from macrophages. J Exp Med. 1992 Aug 1;176(2):599–604. doi: 10.1084/jem.176.2.599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Daff S., Sagami I., Shimizu T. The 42-amino acid insert in the FMN domain of neuronal nitric-oxide synthase exerts control over Ca(2+)/calmodulin-dependent electron transfer. J Biol Chem. 1999 Oct 22;274(43):30589–30595. doi: 10.1074/jbc.274.43.30589. [DOI] [PubMed] [Google Scholar]
  9. Gachhui R., Presta A., Bentley D. F., Abu-Soud H. M., McArthur R., Brudvig G., Ghosh D. K., Stuehr D. J. Characterization of the reductase domain of rat neuronal nitric oxide synthase generated in the methylotrophic yeast Pichia pastoris. Calmodulin response is complete within the reductase domain itself. J Biol Chem. 1996 Aug 23;271(34):20594–20602. doi: 10.1074/jbc.271.34.20594. [DOI] [PubMed] [Google Scholar]
  10. Garthwaite J., Charles S. L., Chess-Williams R. Endothelium-derived relaxing factor release on activation of NMDA receptors suggests role as intercellular messenger in the brain. Nature. 1988 Nov 24;336(6197):385–388. doi: 10.1038/336385a0. [DOI] [PubMed] [Google Scholar]
  11. Griffith O. W., Stuehr D. J. Nitric oxide synthases: properties and catalytic mechanism. Annu Rev Physiol. 1995;57:707–736. doi: 10.1146/annurev.ph.57.030195.003423. [DOI] [PubMed] [Google Scholar]
  12. Gutierrez A., Doehr O., Paine M., Wolf C. R., Scrutton N. S., Roberts G. C. Trp-676 facilitates nicotinamide coenzyme exchange in the reductive half-reaction of human cytochrome P450 reductase: properties of the soluble W676H and W676A mutant reductases. Biochemistry. 2000 Dec 26;39(51):15990–15999. doi: 10.1021/bi002135n. [DOI] [PubMed] [Google Scholar]
  13. Gutierrez A., Lian L. Y., Wolf C. R., Scrutton N. S., Roberts G. C. Stopped-flow kinetic studies of flavin reduction in human cytochrome P450 reductase and its component domains. Biochemistry. 2001 Feb 20;40(7):1964–1975. doi: 10.1021/bi001719m. [DOI] [PubMed] [Google Scholar]
  14. Ignarro L. J., Buga G. M., Wood K. S., Byrns R. E., Chaudhuri G. Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide. Proc Natl Acad Sci U S A. 1987 Dec;84(24):9265–9269. doi: 10.1073/pnas.84.24.9265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Klatt P., Schmidt K., Mayer B. Brain nitric oxide synthase is a haemoprotein. Biochem J. 1992 Nov 15;288(Pt 1):15–17. doi: 10.1042/bj2880015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Leclerc D., Wilson A., Dumas R., Gafuik C., Song D., Watkins D., Heng H. H., Rommens J. M., Scherer S. W., Rosenblatt D. S. Cloning and mapping of a cDNA for methionine synthase reductase, a flavoprotein defective in patients with homocystinuria. Proc Natl Acad Sci U S A. 1998 Mar 17;95(6):3059–3064. doi: 10.1073/pnas.95.6.3059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Marletta M. A. Nitric oxide synthase structure and mechanism. J Biol Chem. 1993 Jun 15;268(17):12231–12234. [PubMed] [Google Scholar]
  18. Matsuda H., Iyanagi T. Calmodulin activates intramolecular electron transfer between the two flavins of neuronal nitric oxide synthase flavin domain. Biochim Biophys Acta. 1999 Dec 27;1473(2-3):345–355. doi: 10.1016/s0304-4165(99)00193-2. [DOI] [PubMed] [Google Scholar]
  19. McMillan K., Masters B. S. Prokaryotic expression of the heme- and flavin-binding domains of rat neuronal nitric oxide synthase as distinct polypeptides: identification of the heme-binding proximal thiolate ligand as cysteine-415. Biochemistry. 1995 Mar 21;34(11):3686–3693. doi: 10.1021/bi00011a025. [DOI] [PubMed] [Google Scholar]
  20. Miller R. T., Martásek P., Omura T., Siler Masters B. S. Rapid kinetic studies of electron transfer in the three isoforms of nitric oxide synthase. Biochem Biophys Res Commun. 1999 Nov;265(1):184–188. doi: 10.1006/bbrc.1999.1643. [DOI] [PubMed] [Google Scholar]
  21. Miller R. T., Martásek P., Roman L. J., Nishimura J. S., Masters B. S. Involvement of the reductase domain of neuronal nitric oxide synthase in superoxide anion production. Biochemistry. 1997 Dec 9;36(49):15277–15284. doi: 10.1021/bi972022c. [DOI] [PubMed] [Google Scholar]
  22. Munro A. W., Noble M. A., Robledo L., Daff S. N., Chapman S. K. Determination of the redox properties of human NADPH-cytochrome P450 reductase. Biochemistry. 2001 Feb 20;40(7):1956–1963. doi: 10.1021/bi001718u. [DOI] [PubMed] [Google Scholar]
  23. Murataliev M. B., Feyereisen R. Interaction of NADP(H) with oxidized and reduced P450 reductase during catalysis. Studies with nucleotide analogues. Biochemistry. 2000 May 2;39(17):5066–5074. doi: 10.1021/bi992917k. [DOI] [PubMed] [Google Scholar]
  24. Noble M. A., Munro A. W., Rivers S. L., Robledo L., Daff S. N., Yellowlees L. J., Shimizu T., Sagami I., Guillemette J. G., Chapman S. K. Potentiometric analysis of the flavin cofactors of neuronal nitric oxide synthase. Biochemistry. 1999 Dec 14;38(50):16413–16418. doi: 10.1021/bi992150w. [DOI] [PubMed] [Google Scholar]
  25. Oprian D. D., Coon M. J. Oxidation-reduction states of FMN and FAD in NADPH-cytochrome P-450 reductase during reduction by NADPH. J Biol Chem. 1982 Aug 10;257(15):8935–8944. [PubMed] [Google Scholar]
  26. Paine M. J., Garner A. P., Powell D., Sibbald J., Sales M., Pratt N., Smith T., Tew D. G., Wolf C. R. Cloning and characterization of a novel human dual flavin reductase. J Biol Chem. 2000 Jan 14;275(2):1471–1478. doi: 10.1074/jbc.275.2.1471. [DOI] [PubMed] [Google Scholar]
  27. Palmer R. M., Ferrige A. G., Moncada S. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature. 1987 Jun 11;327(6122):524–526. doi: 10.1038/327524a0. [DOI] [PubMed] [Google Scholar]
  28. Pollock V. V., Barber M. J. Kinetic and mechanistic properties of biotin sulfoxide reductase. Biochemistry. 2001 Feb 6;40(5):1430–1440. doi: 10.1021/bi001842d. [DOI] [PubMed] [Google Scholar]
  29. Sem D. S., Kasper C. B. Effect of ionic strength on the kinetic mechanism and relative rate limitation of steps in the model NADPH-cytochrome P450 oxidoreductase reaction with cytochrome c. Biochemistry. 1995 Oct 3;34(39):12768–12774. doi: 10.1021/bi00039a037. [DOI] [PubMed] [Google Scholar]
  30. Sem D. S., Kasper C. B. Kinetic mechanism for the model reaction of NADPH-cytochrome P450 oxidoreductase with cytochrome c. Biochemistry. 1994 Oct 11;33(40):12012–12021. doi: 10.1021/bi00206a002. [DOI] [PubMed] [Google Scholar]
  31. Sheta E. A., McMillan K., Masters B. S. Evidence for a bidomain structure of constitutive cerebellar nitric oxide synthase. J Biol Chem. 1994 May 27;269(21):15147–15153. [PubMed] [Google Scholar]
  32. Stuehr D. J. Structure-function aspects in the nitric oxide synthases. Annu Rev Pharmacol Toxicol. 1997;37:339–359. doi: 10.1146/annurev.pharmtox.37.1.339. [DOI] [PubMed] [Google Scholar]
  33. Viola R. E., Cook P. F., Cleland W. W. Stereoselective preparation of deuterated reduced nicotinamide adenine nucleotides and substrates by enzymatic synthesis. Anal Biochem. 1979 Jul 15;96(2):334–340. doi: 10.1016/0003-2697(79)90590-6. [DOI] [PubMed] [Google Scholar]
  34. Wang M., Roberts D. L., Paschke R., Shea T. M., Masters B. S., Kim J. J. Three-dimensional structure of NADPH-cytochrome P450 reductase: prototype for FMN- and FAD-containing enzymes. Proc Natl Acad Sci U S A. 1997 Aug 5;94(16):8411–8416. doi: 10.1073/pnas.94.16.8411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Wolthers K. R., Schimerlik M. I. Reaction of neuronal nitric-oxide synthase with 2,6-dichloroindolphenol and cytochrome c3+: influence of the electron acceptor and binding of Ca2+-activated calmodulin on the kinetic mechanism. Biochemistry. 2001 Apr 17;40(15):4722–4737. doi: 10.1021/bi0023495. [DOI] [PubMed] [Google Scholar]
  36. Wolthers Kirsten R., Schimerlik Michael I. Neuronal nitric oxide synthase: substrate and solvent kinetic isotope effects on the steady-state kinetic parameters for the reduction of 2,6-dichloroindophenol and cytochrome c(3+). Biochemistry. 2002 Jan 8;41(1):196–204. doi: 10.1021/bi0109461. [DOI] [PubMed] [Google Scholar]
  37. Zhang J., Martàsek P., Paschke R., Shea T., Siler Masters B. S., Kim J. J. Crystal structure of the FAD/NADPH-binding domain of rat neuronal nitric-oxide synthase. Comparisons with NADPH-cytochrome P450 oxidoreductase. J Biol Chem. 2001 Jul 25;276(40):37506–37513. doi: 10.1074/jbc.M105503200. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES