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J. Biol. Chem., Vol. 279, Issue 25, 26489-26499, June 18, 2004

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Heme Distortion Modulated by Ligand-Protein Interactions in Inducible Nitric-oxide Synthase^*

David Li, Dennis J. Stuehr¶, Syun-Ru Yeh, and Denis L. Rousseau||

From the Department of Physiology and Biophysics, Albert Einstein College of Medicine of Yeshiva University, Bronx, New York 10461 and the ^¶Department of Immunology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195

The catalytic center of nitric-oxide synthase (NOS) consists of a thiolate-coordinated heme macrocycle, a tetrahydrobiopterin (H4B) cofactor, and an L-arginine (L-Arg)/N-hydroxyarginine substrate binding site. To determine how the interplay between the cofactor, the substrates, and the protein matrix housing the heme regulates the enzymatic activity of NOS, the CO-, NO-, and CN^--bound adducts of the oxygenase domain of the inducible isoform of NOS (iNOS_oxy) were examined with resonance Raman spectroscopy. The Raman data of the CO-bound ferrous protein demonstrated that the presence of L-Arg causes the Fe–C–O moiety to adopt a bent structure because of an H-bonding interaction whereas H4B binding exerts no effect. Similar behavior was found in the CN^--bound ferric protein and in the nitric oxide (NO)-bound ferrous protein. In contrast, in the NO-bound ferric complexes, the addition of L-Arg alone does not affect the structural properties of the Fe–N–O moiety, but H4B binding forces it to adopt a bent structure, which is further enhanced by the subsequent addition of L-Arg. The differential interactions between the various heme ligands and the protein matrix in response to L-Arg and/or H4B binding is coupled to heme distortions, as reflected by the development of a variety of out-of-plane heme modes in the low frequency Raman spectra. The extent and symmetry of heme deformation modulated by ligand, substrate, and cofactor binding may provide important control over the catalytic and autoinhibitory properties of the enzyme.

Received for publication, January 28, 2004 , and in revised form, March 11, 2004.

^* This work was supported by Grants CA53914 and GM51491 (to D. J. S.), HL65465 (to S.-R. Y.), and GM54806 (to D. L. R.) from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by the Medical Scientist Training Program (GM07288) and the Molecular Biophysics Training Grant GM08572 at Albert Einstein College of Medicine.

^|| To whom correspondence should be addressed. Tel.: 718-430-4264; Fax: 718-430-8808; E-mail: rousseau{at}aecom.yu.edu.

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