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  • Title: Interactions between substrate analogues and heme ligands in nitric oxide synthase.
    Author: Wang J, Stuehr DJ, Rousseau DL.
    Journal: Biochemistry; 1997 Apr 15; 36(15):4595-606. PubMed ID: 9109669.
    Abstract:
    The substrate binding site in nitric oxide synthase (NOS) can accommodate the physiological substrates, L-arginine and N(omega)-hydroxy L-arginine as well as many substrate analogues and inhibitors. Resonance Raman spectra of carbon monoxide-bound NOS were measured to determine how these substrates and analogues interact with heme, the prosthetic group which activates oxygen for the catalytic generation of NO and citrulline from arginine in the enzyme. Two distinct conformations of the Fe-C-O moiety were detected in the resonance Raman spectra, although in the optical absorption spectra the two species are indistinguishable. In one, termed the beta-form, the Fe-CO stretching frequency and the C-O stretching frequency, located at approximately 487 and approximately 1949 cm(-1), respectively, demonstrate that the Fe-C-O group adopts a linear conformation perpendicular to the heme plane ("open" structure). In the other, termed the alpha-form, frequencies of approximately 502 and approximately 1929 cm(-1), respectively, indicate that the binding properties of bound CO are significantly affected by its immediate environment thereby leading to a "closed" structure. In the presence of L-arginine or N(omega)-OH-L-arginine all of the molecules exhibit the closed structure, indicating that the substrates exert a strong polar (and/or steric) effect on the heme-bound ligand. In the absence of any substrate or inhibitor only half of the heme population adopts the open structure whereas the rest of the heme content retains the closed conformation. It is proposed that in this population with the closed structure tetrahydrobiopterin, a cofactor of NO synthase, may reside in close proximity to the heme-bound ligand and interact with it in a similar manner as do substrates. The inverse correlation between the Fe-CO and C-O stretching modes suggests that in NOS the bonding of the cysteine to the heme iron may be weaker, as found in chloroperoxidase, than in cytochrome P-450 enzymes. This work continually proves resonance Raman spectroscopy as a powerful probe for the interactions between substrate/inhibitor and the heme active site of proteins.
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