Role of substrate functional groups in binding to nitric oxide synthase.

The interactions between the heme CO ligand in the oxygenase domain of nitric oxide synthase and a set of substrate analogues were determined by measuring the resonance Raman spectra of the Fe-C-O vibrational modes. Substrates were selected that have variations in all the functional units: the guanidino group, the amino acid site and the number of methylene units connecting the two ends. In comparison to the substrate free form of the enzyme, Interactions of the analogues with the CO moiety caused the Fe-CO stretching and the Fe-C-O bending modes to shift in frequency due to the electrostatic environment. An unmodified guanidino group interacted with the CO in a similar fashion despite changes in the amino acid end. However, an unmodified amino acid end is required for catalysis owing to the H-bonding network involving the substrate, the heme and the pterin cofactor.