Arg375 Tunes Tetrahydrobiopterin Functions and Modulates Catalysis by Inducible Nitric Oxide Synthase

Overview

Journal J Inorg Biochem

Specialty Biochemistry

Date 2011 Dec 17

PMID 22173094

Citations 6

Authors

Zhi-Qiang Wang

Jesus Tejero

Chin-Chuan Wei

Mohammad Mahfuzul Haque

Jerome Santolini

Mohammed Fadlalla

Ashis Biswas

Dennis J Stuehr

Affiliations

Soon will be listed here.

Abstract

NO synthase enzymes (NOS) support unique single-electron transitions of a bound H(4)B cofactor during catalysis. Previous studies showed that both the pterin structure and surrounding protein residues impact H(4)B redox function during catalysis. A conserved Arg residue (Arg375 in iNOS) forms hydrogen bonds with the H(4)B ring. In order to understand the role of this residue in modulating the function of H(4)B and overall NO synthesis of the enzyme, we generated and characterized three mutants R375D, R375K and R375N of the oxygenase domain of inducible NOS (iNOSoxy). The mutations affected the dimer stability of iNOSoxy and its binding affinity toward substrates and H(4)B to varying degrees. Optical spectra of the ferric, ferrous, ferrous dioxy, ferrous-NO, ferric-NO, and ferrous-CO forms of each mutant were similar to the wild-type. However, mutants displayed somewhat lower heme midpoint potentials and faster ferrous heme-NO complex reactivity with O(2). Unlike the wild-type protein, mutants could not oxidize NOHA to nitrite in a H(2)O(2)-driven reaction. Mutation could potentially change the ferrous dioxy decay rate, H(4)B radical formation rate, and the amount of the Arg hydroxylation during single turnover Arg hydroxylation reaction. All mutants were able to form heterodimers with the iNOS G450A full-length protein and displayed lower NO synthesis activities and uncoupled NADPH consumption. We conclude that the conserved residue Arg375 (1) regulates the tempo and extent of the electron transfer between H(4)B and ferrous dioxy species and (2) controls the reactivity of the heme-based oxidant formed after electron transfer from H(4)B during steady state NO synthesis and H(2)O(2)-driven NOHA oxidation. Thus, Arg375 modulates the redox function of H(4)B and is important in controlling the catalytic function of NOS enzymes.

Citing Articles

Spotlight on ROS and 3-Adrenoreceptors Fighting in Cancer Cells.

Calvani M, Subbiani A, Vignoli M, Favre C Oxid Med Cell Longev. 2020; 2019:6346529.

PMID: 31934266 PMC: 6942895. DOI: 10.1155/2019/6346529.

Hydroxyl Radical-Coupled Electron-Transfer Mechanism of Flavin-Dependent Hydroxylases.

Tweedy S, Benitez A, Narayan A, Zimmerman P, Brooks 3rd C, Wymore T J Phys Chem B. 2019; 123(38):8065-8073.

PMID: 31532200 PMC: 6943927. DOI: 10.1021/acs.jpcb.9b08178.

Inducible nitric oxide synthase: Regulation, structure, and inhibition.

Cinelli M, Do H, Miley G, Silverman R Med Res Rev. 2019; 40(1):158-189.

PMID: 31192483 PMC: 6908786. DOI: 10.1002/med.21599.

Optimization of Blood-Brain Barrier Permeability with Potent and Selective Human Neuronal Nitric Oxide Synthase Inhibitors Having a 2-Aminopyridine Scaffold.

Do H, Li H, Chreifi G, Poulos T, Silverman R J Med Chem. 2019; 62(5):2690-2707.

PMID: 30802056 PMC: 6586428. DOI: 10.1021/acs.jmedchem.8b02032.

Phosphorylation inactivation of endothelial nitric oxide synthesis in pulmonary arterial hypertension.

Ghosh S, Gupta M, Xu W, Mavrakis D, Janocha A, Comhair S Am J Physiol Lung Cell Mol Physiol. 2016; 310(11):L1199-205.

PMID: 27130529 PMC: 4935470. DOI: 10.1152/ajplung.00092.2016.

References

Masters B, McMillan K, Sheta E, NISHIMURA J, Roman L, Martasek P . Neuronal nitric oxide synthase, a modular enzyme formed by convergent evolution: structure studies of a cysteine thiolate-liganded heme protein that hydroxylates L-arginine to produce NO. as a cellular signal. FASEB J. 1996; 10(5):552-8. DOI: 10.1096/fasebj.10.5.8621055. View

Gorren A, List B, Schrammel A, Pitters E, Hemmens B, Werner E . Tetrahydrobiopterin-free neuronal nitric oxide synthase: evidence for two identical highly anticooperative pteridine binding sites. Biochemistry. 1996; 35(51):16735-45. DOI: 10.1021/bi961931j. View

Tejero J, Santolini J, Stuehr D . Fast ferrous heme-NO oxidation in nitric oxide synthases. FEBS J. 2009; 276(16):4505-14. PMC: 2737443. DOI: 10.1111/j.1742-4658.2009.07157.x. View

Wei C, Crane B, Stuehr D . Tetrahydrobiopterin radical enzymology. Chem Rev. 2003; 103(6):2365-83. DOI: 10.1021/cr0204350. View

Adak S, Wang Q, Stuehr D . Molecular basis for hyperactivity in tryptophan 409 mutants of neuronal NO synthase. J Biol Chem. 2000; 275(23):17434-9. DOI: 10.1074/jbc.M000846200. View

Abu-Soud H, Wu C, Ghosh D, Stuehr D . Stopped-flow analysis of CO and NO binding to inducible nitric oxide synthase. Biochemistry. 1998; 37(11):3777-86. DOI: 10.1021/bi972398q. View

Schmidt P, Lange R, Gorren A, Werner E, Mayer B, Andersson K . Formation of a protonated trihydrobiopterin radical cation in the first reaction cycle of neuronal and endothelial nitric oxide synthase detected by electron paramagnetic resonance spectroscopy. J Biol Inorg Chem. 2001; 6(2):151-8. DOI: 10.1007/s007750000185. View

Chen P, Berka V, Wu K . Differential effects of mutations in human endothelial nitric oxide synthase at residues Tyr-357 and Arg-365 on L-arginine hydroxylation and GN-hydroxy-L-arginine oxidation. Arch Biochem Biophys. 2003; 411(1):83-92. DOI: 10.1016/s0003-9861(02)00729-4. View

Tejero J, Biswas A, Wang Z, Page R, Haque M, Hemann C . Stabilization and characterization of a heme-oxy reaction intermediate in inducible nitric-oxide synthase. J Biol Chem. 2008; 283(48):33498-507. PMC: 2586280. DOI: 10.1074/jbc.M806122200. View

10.

Sorlie M, Gorren A, Marchal S, Shimizu T, Lange R, Andersson K . Single-turnover of nitric-oxide synthase in the presence of 4-amino-tetrahydrobiopterin: proposed role for tetrahydrobiopterin as a proton donor. J Biol Chem. 2003; 278(49):48602-10. DOI: 10.1074/jbc.M305682200. View

11.

Stuehr D . Spectral characterization of brain and macrophage nitric oxide synthases. Cytochrome P-450-like hemeproteins that contain a flavin semiquinone radical. J Biol Chem. 1992; 267(29):20547-50. View

12.

Rusche K, Spiering M, Marletta M . Reactions catalyzed by tetrahydrobiopterin-free nitric oxide synthase. Biochemistry. 1998; 37(44):15503-12. DOI: 10.1021/bi9813936. View

13.

Stuehr D . Mammalian nitric oxide synthases. Biochim Biophys Acta. 1999; 1411(2-3):217-30. DOI: 10.1016/s0005-2728(99)00016-x. View

14.

Pufahl R, Wishnok J, Marletta M . Hydrogen peroxide-supported oxidation of NG-hydroxy-L-arginine by nitric oxide synthase. Biochemistry. 1995; 34(6):1930-41. DOI: 10.1021/bi00006a014. View

15.

Wang Z, Wei C, Santolini J, Panda K, Wang Q, Stuehr D . A tryptophan that modulates tetrahydrobiopterin-dependent electron transfer in nitric oxide synthase regulates enzyme catalysis by additional mechanisms. Biochemistry. 2005; 44(12):4676-90. DOI: 10.1021/bi047508p. View

16.

Wang Z, Wei C, Sharma M, Pant K, Crane B, Stuehr D . A conserved Val to Ile switch near the heme pocket of animal and bacterial nitric-oxide synthases helps determine their distinct catalytic profiles. J Biol Chem. 2004; 279(18):19018-25. DOI: 10.1074/jbc.M311663200. View

17.

Nathan C, Shiloh M . Reactive oxygen and nitrogen intermediates in the relationship between mammalian hosts and microbial pathogens. Proc Natl Acad Sci U S A. 2000; 97(16):8841-8. PMC: 34021. DOI: 10.1073/pnas.97.16.8841. View

18.

Tejero J, Biswas A, Haque M, Wang Z, Hemann C, Varnado C . Mesohaem substitution reveals how haem electronic properties can influence the kinetic and catalytic parameters of neuronal NO synthase. Biochem J. 2010; 433(1):163-74. DOI: 10.1042/BJ20101353. View

19.

Sagami I, Sato Y, Daff S, Shimizu T . Aromatic residues and neighboring Arg414 in the (6R)-5,6,7, 8-tetrahydro-L-biopterin binding site of full-length neuronal nitric-oxide synthase are crucial in catalysis and heme reduction with NADPH. J Biol Chem. 2000; 275(34):26150-7. DOI: 10.1074/jbc.M000534200. View

20.

Wang Z, Wei C, Ghosh S, Meade A, Hemann C, Hille R . A conserved tryptophan in nitric oxide synthase regulates heme-dioxy reduction by tetrahydrobiopterin. Biochemistry. 2001; 40(43):12819-25. DOI: 10.1021/bi011182s. View