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NOS vs NOX NCI workshop

NOS vs NOX NCI workshop. Dr. Dennis Stuehr, Lerner Research Institute, Cleveland Clinic. Lerner Research Institute, Cleveland Clinic. GOAL: Discuss some insights provided by the NOS regarding structure-function of redox enzymes.

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NOS vs NOX NCI workshop

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  1. NOS vs NOXNCI workshop Dr. Dennis Stuehr, Lerner Research Institute, Cleveland Clinic

  2. Lerner Research Institute, Cleveland Clinic

  3. GOAL: Discuss some insights provided by the NOS regarding structure-function of redox enzymes Some structural and catalytic features of NOS enzymes. Compare/contrast to cytP450 system and NOX. One topic of special interest: Mechanisms and regulation of NOS electron transfer.

  4. Nitric oxide synthases • In Animals- As many as three structurally-similar isozymes, plus splice variants. • In Bacteria- Heme proteins that are structurally similar to animal NOS, but do not have an attached flavoprotein domain. • In Plants- DNA sequences predict structurally-distinct NOS enzymes.

  5. Two-step oxidation catalyzed by the NO synthases This is a novel biological oxidation of Arginine

  6. Some biological fates of NO

  7. NO synthases are the only enzymes known to utilize these four redox cofactors

  8. Electron transfer pathway in a NOS dimer

  9. Ferredoxin NADP+ reductase module A common path of electron transfer in nNOS FMN module I I I I I I I I I I I I 1 2 3 Other NADPH-dependent dual flavin reductases Cytochrome P450 reductase Novel reductase-1 Sulfite reductase Cytochrome P450BM3 Methionine synthase reductase

  10. Stepwise mechanism for NOS dimer assembly in animal cells

  11. iNOSoxy dimer

  12. Extensive dimer interface in NOSoxy

  13. d114iNOSox monomer (thin) iNOSox dimer + H4B/Arg (thick) Dimerization & Arg/H4B binding are associated with structural change at the NOS dimer interface a7A HL a9

  14. NOS is a heme-thiolate enzyme that requires two electrons to activate O2 Oxygen activation mechanism in NOS is similar to that in cyt P450

  15. HuP450 P450BM3 iNOS oxy domain P450nor Unique fold, Less helical

  16. Heme flipped, loop longer, helix downstream

  17. Heme environment in NOSFew Proton-donating AA in distal pocket Extensive H-bonding with cys thiolate heme ligand

  18. Structures of the heme environment on the proximal side of cysteine-coordinated heme proteins Couture, M. et al. J. Biol. Chem. 2001;276:38280-38288

  19. iNOSoxy Dimer Core structure Core structure N-terminal hook elements

  20. H4B is surrounded by residues from both subunits of the dimer

  21. SUMMARY: Some unique features of the NOS oxygenase domain • Extensive Beta sheet structure with fewer helical elements than in P450 • Less polar distal heme pocket • Greater hydrogen bonding to heme thiolate ligand • Extensive dimer interface with integrated H4B binding sites

  22. Redox functions of H4B and related pterins in select enzymes

  23. University of Leicester APX/ascorbate complex (no substrate on the left, substrate bound on the right) C32 K30 K30 g C32 d R172 Ascorbate R172 Sharp, Mewies, Moody and Raven, Nature Struct. Biol. (2003) 10, 303.

  24. NOS oxygen activation in relation to electron import, substrate oxidation, and superoxide or H2O2 release

  25. Rapid mixing to study a single catalytic turnover in NOS

  26. Heme FeIIO2 formation and decay, H4B radical formation and decay, Arg hydroxylation, and heme FeIII recovery in a single-turnover Arg reaction catalyzed by iNOSoxy at 10 C. After O2 binds, the electron transfer from H4B is rate- limiting for the reaction

  27. Why do NOS use H4B as a source of the 2nd electron? kinetic partitioning is critical Ans1. Because H4B reduces the heme-dioxy intermediate faster than can the flavoprotein domain.

  28. Q. What controls the rate of H4B radical formation in NOS?A. The structure of H4B itself, and the surrounding protein residues.

  29. Compare Tetrahydrobiopterin with its 5-Methyl derivative Does the structure of H4B influence its electron transfer?

  30. How do the surrounding protein residues influence H4B electron transfer?

  31. There is a good correlation between the biopterin radical formation rate and resultant radical stability in NOS 5MeH4B/nNOSoxy 5MeH4B/iNOSoxy Biopterin radical formation (s-1) 5MeH4B/eNOSoxy H4B/nNOSoxy H4B/eNOSoxy H4B/iNOSoxy H4B/W457F H4B/W457A t1/2 (s) Wei, et al. Biochemistry (2003) 42, 1969

  32. Cation-Pi interaction helps determine the biopterin radical stability cation H4B+ pi-electron W457 indole ring Dougherty, D. A. (1996) Science 271 , 163-168 electrostatic potential surface Ala Phe Trp Wei, et al. Chem. Rev. (2003) 103, 2365

  33. NOS Flavoprotein domain:How do electrons transfer to the heme in NOS? How is the process regulated?

  34. Large subdomain movements occur during electron transfer

  35. Three Flavin Redox States are involved during e transfer

  36. Structure of the nNOS Flavoprotein (nNOSr)Garcin et al, JBC 2004 Regulatory elements visualized!

  37. Proposed holo-NOS assembly and domain movements Garcin, E. D. et al. J. Biol. Chem. 2004;279:37918-37927

  38. Regulation of NOS electron transfer- 1. Electron transfer into and out of the NOS flavoprotein domain is repressed relative to the other related flavoproteins. 2. Calmodulin binding relieves the repression. (This is a novel function for a calcium binding protein.)

  39. Comparative reductase activities of NOS and some related flavoproteins

  40. Hypothesis:A conformational equilibrium controls the electron transfer reactions of the FMN module

  41. Hypothesis Both common and unique structural elements regulate electron transfer by the NOS flavoprotein. Repression of NOS Electron transfer, and its relief by CaM, involves the unique regulatory elements in the flavoprotein domain.

  42. Electronegative surface charges on the NOS FMN module

  43. Complementary surface charges on the NADPH-FAD domain

  44. Unique structural elements in the NOS flavoprotein * • Salerno, et. al. (1997) J. Biol. Chem.272, 29769-29777. • Daff, S., Sagami, I., & Shimizu, T. (1999) J. Biol. Chem.274, 30589-30595. • Roman, et. al. (2000) J. Biol. Chem.275, 29225-29232. • Montgomery, H. J., Romanov, V., & Guillemette, J. G. (2000) J. Biol. Chem. 275, 5052-5058. • Roman, et. al. (2000) J. Biol. Chem. 275, 21914-21919. • Nishida, C. R., & Ortiz de Montellano, P. R. (1999) J Biol Chem. 274, 14692-14698. • Chen, P. F., & Wu, K. K. (2000) J. Biol. Chem.275, 13155-13163. • Lane, P., & Gross, S. S. (2002) J. Biol. Chem. In press.

  45. Structure of the nNOS Flavoprotein (nNOSr)Garcin et al, JBC 2004 Regulatory elements visualized!

  46. Hypotheses derived from the literature The FMN-shielded conformation is favored in Cam-free NOSr. NADP(H) binding is required to stabilize the FMN-shielded conformation in CaM-free NOSr. CaM binding favors the FMN deshielded conformation. Daff et al, (2002) JBC 277, 33987-94

  47. nNOSr Conformational Equilibrium

  48. Does our structure show the nNOS Flavoprotein in the FMN-locked conformation??

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