INTERACTIONS BETWEEN SUBSTRATE ANALOGUES AND HEME LIGANDS IN NITRIC OXIDE SYNTHASE

The substrate binding site in nitric oxide synthase (NOS) can accommodate the physiological substrates, l-arginine and Nω-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 β-form, the Fe−CO stretching frequency and the C−O stretching frequency, located at ∼487 and ∼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 α-form, frequencies of ∼502 and ∼1929 cm-1, respectively, indicate that the binding properties of bound CO are sign...