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J. Biol. Chem., Vol. 276, Issue 41, 38280-38288, October 12, 2001
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From the Nitric-oxide synthase (NOS) catalyzes the
formation of NO and citrulline from L-arginine and
oxygen. However, the NO so formed has been found to auto-inhibit the
enzymatic activity significantly. We hypothesized that the NO
reactivity is in part controlled by hydrogen bonding between the
conserved tryptophan residue (position 409 in the neuronal isoform of
NOS (nNOS)) and the cysteine residue that forms the proximal bond to
the heme. By using resonance Raman spectroscopy and NO as a probe of
the heme environment, we show that in the W409F and W409Y mutants of
the oxygenase domain of the neuronal enzyme (nNOSox), the Fe-NO bond in
the Fe3+NO complex is weaker than in the wild type enzyme,
consistent with the loss of a hydrogen bond on the sulfur atom of the
proximal cysteine residue. The weaker Fe-NO bond in the W409F and W409Y mutants might result in a faster rate of NO dissociation from the
ferric heme in the Trp-409 mutants as compared with the wild type
enzyme, which could contribute to the lower accumulation of the
inhibitory NO-bound complexes observed during catalysis with the
Trp-409 mutants (Adak, S., Crooks, C., Wang, Q., Crane, B. R.,
Tainer, J. A., Getzoff, E. D., and Stuehr, D. J. (1999) J. Biol. Chem. 274, 26907-26911). The optical and
resonance Raman spectra of the Fe2+NO complexes of the
Trp-409 mutants differ from those of the wild type enzyme and indicate
that a significant population of a five-coordinate Fe2+NO
complex is present. These data show that the hydrogen bond provided by
the Trp-409 residue is necessary to maintain the thiolate coordination
when NO binds to the ferrous heme. Taken together our results indicate
that the heme environment on the proximal side of nNOS is critical for
the formation of a stable iron-cysteine bond and for the control of the
electronic properties of heme-NO complexes.
Regulation of the Properties of the Heme-NO Complexes
in Nitric-oxide Synthase by Hydrogen Bonding to the Proximal
Cysteine*
,¶
Department of Physiology and Biophysics,
Albert Einstein College of Medicine, Bronx, New York 10461 and the
§ Department of Immunology, Lerner Research Institute,
Cleveland Clinic Foundation, Cleveland, Ohio 44195
*
This work was supported by National Institutes of Health
Grants GM54806 and GM54812 (to D. L. R.) and CA53914 (to D. J. S.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
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