Interactions between Substrate Analogues and Heme Ligands in Nitric Oxide Synthase

Abstract

The substrate binding site in nitric oxide synthase (NOS) can accommodate the physiological substrates, L-arginine and N ω -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 R-form, frequencies of ∼502 and ∼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 ω -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 hemebound 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.

The Ferrous Dioxygen Complex of the Oxygenase Domain of Neuronal Nitric-oxide Synthase

“…Both the substrate and H 4 B molecules participate in an extensive hydrogen bond network that includes a heme propionate, water molecules, and several amino acids. The proximity of the L-arginine binding site to the heme is consistent with the interactions of substrates with heme-bound ligands such as CO, NO, and imidazole, which were revealed by optical (19), EPR (20), and resonance Raman spectroscopies (21)(22)(23). In addition, the substrates and H 4 B, when present, cause a shift in the spin state of the ferric enzyme (24).…”

“…Crystallographic data indicate that in iNOSoxy and chloroperoxidase, the proximal cysteine is involved in additional hydrogen bonds with surrounding residues as compared with cytochrome P450 (35,36). This observation is consistent with resonance Raman studies of the CO complexes of nNOS and chloroperoxidase, which indicate that the cysteine-iron linkage is weaker in these proteins than in cytochrome P450 (21). Thus the slightly lower frequency of the O-O line in nNOSoxy may result from a weaker iron-cysteine bond as compared with that of cytochrome P450.…”

“…Last, we cannot exclude the possibility that in substrate-free Fe 2ϩ (O 2 ) nNOSoxy, the dioxygen molecule is involved in steric/polar interactions with a heme pocket residue. This proposition could account for the fact that in substrate-free CO nNOS (21) and CO nNOSoxy (Fig. 4c), approximately half of the enzyme adopts the closed conformation with the Fe-CO line at 502 cm…”

“…In CO-nNOS, it was shown that the heme pocket undergoes a conformational change upon binding of L-arginine that results in a shift of the Fe-CO line from 487/501 to 503 cm Ϫ1 , a shift of the ␦ Fe-CO line from 562 to 566 cm Ϫ1 , and a shift of the C-O line from 1930/1949 to 1929 cm Ϫ1 (21,23). In the L-arginine-bound conformation, it was concluded that the heme pocket adopts a closed structure, where the substrate exerts a strong polar and/or steric effect on the heme-bound CO molecule.…”

Raman Evidence for Specific Substrate-Induced Structural Changes in the Heme Pocket of Human Cytochrome P450 Aromatase during the Three Consecutive Oxygen Activation Steps

“…The addition of substrates and analogue dramatically alters the peak positions and shapes for the Raman lines associated with CO as observed for other P450s (34,41,42) and NOSs (43,44). The ν Fe-CO line was shifted to 485 (for AD-bound P450arom) and 484 cm -1 (for Test-bound P450arom), and the δ FeCO line was intensified by the addition of AD or Test [ Figure 5A(c,e)].…”