Heme distortion modulated by ligand-protein interactions in inducible nitric-oxide synthase.
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.
Li
David
D
Department of Physiology and Biophysics, Albert Einstein College of Medicine of Yeshiva University, Bronx, New York 10461, USA.
Stuehr
Dennis J
DJ
Yeh
Syun-Ru
SR
Rousseau
Denis L
DL
D001665:Binding Sites; D002384:Catalysis; D004926:Escherichia coli / Q000378:metabolism; D006418:Heme / Q000737:chemistry*; D007501:Iron / Q000737:chemistry; D008024:Ligands; D009569:Nitric Oxide / Q000737:chemistry / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000737:chemistry* / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D013053:Spectrophotometry; D013059:Spectrum Analysis, Raman
D008024:Ligands; D020033:Protein Isoforms; D009569:Nitric Oxide; D006418:Heme; D007501:Iron; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II