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Characterization of C415 mutants of neuronal nitric oxide synthase.
Biochemistry. 1996 Jun 18; 35(24):7772-80.B

Abstract

Nitric oxide synthase (NOS) catalyzes the oxidation of L-arginine to citrulline and nitric oxide. C415H and C415A mutants of the neuronal isoform of NOS (nNOS) were expressed in a baculovirus system and purified to homogeneity for spectral analysis and activity measurements. UV-visible spectra of each mutant lacked an observable Soret peak, suggesting that neither mutant contained heme. When reduced in the presence of CO, however, a small Soret centered at 417 nm could be detected for the C415H mutant, further supporting the assignment of C415 as the axial ligand to the heme. In addition to a deficiency in bound heme, neither mutant had any detectable bound tetrahydrobiopterin, as compared to wild-type enzyme, which had a ratio of 0.84 mol of bound pteridine:1 mol of nNOS 160 kDa subunit. The C415H mutant contained bound FAD and FMN at levels of 1.0 +/- 0.1 and 0.9 +/- 0.1 mol/mol of nNOS subunit, respectively. UV-visible spectra of both nNOS mutants retained the distinctive absorbance due to tightly associated oxidized flavin prosthetic groups. Further, the spectra suggested the presence of a neutral flavin semiquinone. Ferricyanide oxidation of the C415A mutant yielded a spectrum that was essentially that of oxidized flavin. Ferricyanide titration showed that the C415A mutant contained approximately 1 reducing equiv. Circular dichroism spectra suggested that each mutant was folded properly, in that both spectra were found to be essentially identical to the spectrum of wild-type nNOS. Neither mutant could synthesize nitric oxide, and neither mutant had the ability to oxidize NADPH unless an exogenous electron acceptor was added. The rate of cytochrome c reduction by each mutant was found to be slightly less, but very similar to the rate (approximately 20 mumol mg-1 min-1) observed with wild-type nNOS. In all cases, the rate of cytochrome c reduction increased approximately 15-fold with the addition of calmodulin. Overall, these spectral and activity data suggest that C415 is the axial heme ligand and that a point mutation at C415 prevents binding of heme and tetrahydrobiopterin without interfering with the global folding or the reductase function of nNOS.

Authors+Show Affiliations

Department of Biological Chemistry, School of Medicine, University of Michigan, Ann Arbor 48109-1065, USA.No affiliation info availableNo affiliation info available

Pub Type(s)

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

Language

eng

PubMed ID

8672477

Citation

Richards, M K., et al. "Characterization of C415 Mutants of Neuronal Nitric Oxide Synthase." Biochemistry, vol. 35, no. 24, 1996, pp. 7772-80.
Richards MK, Clague MJ, Marletta MA. Characterization of C415 mutants of neuronal nitric oxide synthase. Biochemistry. 1996;35(24):7772-80.
Richards, M. K., Clague, M. J., & Marletta, M. A. (1996). Characterization of C415 mutants of neuronal nitric oxide synthase. Biochemistry, 35(24), 7772-80.
Richards MK, Clague MJ, Marletta MA. Characterization of C415 Mutants of Neuronal Nitric Oxide Synthase. Biochemistry. 1996 Jun 18;35(24):7772-80. PubMed PMID: 8672477.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Characterization of C415 mutants of neuronal nitric oxide synthase. AU - Richards,M K, AU - Clague,M J, AU - Marletta,M A, PY - 1996/6/18/pubmed PY - 1996/6/18/medline PY - 1996/6/18/entrez SP - 7772 EP - 80 JF - Biochemistry JO - Biochemistry VL - 35 IS - 24 N2 - Nitric oxide synthase (NOS) catalyzes the oxidation of L-arginine to citrulline and nitric oxide. C415H and C415A mutants of the neuronal isoform of NOS (nNOS) were expressed in a baculovirus system and purified to homogeneity for spectral analysis and activity measurements. UV-visible spectra of each mutant lacked an observable Soret peak, suggesting that neither mutant contained heme. When reduced in the presence of CO, however, a small Soret centered at 417 nm could be detected for the C415H mutant, further supporting the assignment of C415 as the axial ligand to the heme. In addition to a deficiency in bound heme, neither mutant had any detectable bound tetrahydrobiopterin, as compared to wild-type enzyme, which had a ratio of 0.84 mol of bound pteridine:1 mol of nNOS 160 kDa subunit. The C415H mutant contained bound FAD and FMN at levels of 1.0 +/- 0.1 and 0.9 +/- 0.1 mol/mol of nNOS subunit, respectively. UV-visible spectra of both nNOS mutants retained the distinctive absorbance due to tightly associated oxidized flavin prosthetic groups. Further, the spectra suggested the presence of a neutral flavin semiquinone. Ferricyanide oxidation of the C415A mutant yielded a spectrum that was essentially that of oxidized flavin. Ferricyanide titration showed that the C415A mutant contained approximately 1 reducing equiv. Circular dichroism spectra suggested that each mutant was folded properly, in that both spectra were found to be essentially identical to the spectrum of wild-type nNOS. Neither mutant could synthesize nitric oxide, and neither mutant had the ability to oxidize NADPH unless an exogenous electron acceptor was added. The rate of cytochrome c reduction by each mutant was found to be slightly less, but very similar to the rate (approximately 20 mumol mg-1 min-1) observed with wild-type nNOS. In all cases, the rate of cytochrome c reduction increased approximately 15-fold with the addition of calmodulin. Overall, these spectral and activity data suggest that C415 is the axial heme ligand and that a point mutation at C415 prevents binding of heme and tetrahydrobiopterin without interfering with the global folding or the reductase function of nNOS. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/8672477/Characterization_of_C415_mutants_of_neuronal_nitric_oxide_synthase_ L2 - https://doi.org/10.1021/bi952582g DB - PRIME DP - Unbound Medicine ER -