Interactions between substrate analogues and heme ligands in nitric oxide synthase

Area of Science:

• Biochemistry
• Enzymology
• Spectroscopy

Background:

• Nitric oxide synthase (NOS) utilizes a heme prosthetic group to catalyze NO production.
• The enzyme's active site accommodates various substrates and analogues, including L-arginine.
• Understanding substrate-heme interactions is crucial for elucidating NOS catalytic mechanisms.

Purpose of the Study:

• To investigate the interaction of substrates and analogues with the heme active site of NOS.
• To characterize the conformational changes in the heme-bound ligand environment using spectroscopy.
• To explore the role of cofactors in modulating heme site structure.

Main Methods:

• Resonance Raman spectroscopy was employed to study carbon monoxide-bound NOS.
• Analysis of Fe-CO and C-O stretching frequencies to determine heme-ligand conformations.
• Comparison of spectra in the presence and absence of substrates and inhibitors.

Main Results:

• Two distinct Fe-C-O conformations, termed "open" (beta-form) and "closed" (alpha-form), were identified.
• L-arginine and N(omega)-hydroxy L-arginine binding induced the "closed" conformation, indicating substrate-induced effects.
• In the absence of ligands, approximately half the heme population adopted the "open" structure, potentially involving the cofactor tetrahydrobiopterin.

Conclusions:

• Substrates exert significant polar and/or steric effects on the heme-bound ligand in NOS.
• Tetrahydrobiopterin may interact with the heme-bound ligand in the absence of substrates, stabilizing the "closed" conformation.
• Resonance Raman spectroscopy is a powerful tool for probing substrate-heme interactions within enzyme active sites.