Nitrosyl−Heme Structures of Bacillus subtilis Nitric Oxide Synthase Have Implications for Understanding Substrate Oxidation^,

Abstract

The crystal structures of nitrosyl-heme complexes of a prokaryotic nitric oxide synthase (NOS) from Bacillus subtilis (bsNOS) reveal changes in active-site hydrogen bonding in the presence of the intermediate N(omega)-hydroxy-l-arginine (NOHA) compared to the substrate l-arginine (l-Arg). Correlating with a Val-to-Ile residue substitution in the bsNOS heme pocket, the Fe(II)-NO complex with both l-Arg and NOHA is more bent than the Fe(II)-NO, l-Arg complex of mammalian eNOS [Li, H., Raman, C. S., Martasek, P., Masters, B. S. S., and Poulos, T. L. (2001) Biochemistry 40, 5399-5406]. Structures of the Fe(III)-NO complex with NOHA show a nearly linear nitrosyl group, and in one subunit, partial nitrosation of bound NOHA. In the Fe(II)-NO complexes, the protonated NOHA N(omega) atom forms a short hydrogen bond with the heme-coordinated NO nitrogen, but active-site water molecules are out of hydrogen bonding range with the distal NO oxygen. In contrast, the l-Arg guanidinium interacts more weakly and equally with both NO atoms, and an active-site water molecule hydrogen bonds to the distal NO oxygen. This difference in hydrogen bonding to the nitrosyl group by the two substrates indicates that interactions provided by NOHA may preferentially stabilize an electrophilic peroxo-heme intermediate in the second step of NOS catalysis.

Role of Arginine Guanidinium Moiety in Nitric-oxide Synthase Mechanism of Oxygen Activation

“…The second step, NOHA 3 citrulline ϩ NO, is supposed to involve the direct reaction of the (hydro)peroxo species on the NOHA hydroxyguanidinium moiety, followed by the rearrangement of the resulting tetrahedral complex and the release of citrulline and NO (26,29). These models are supported by crystallographic, spectroscopic, and cryogenic experimental evidences (39,69,80,83,87), but recently serious questions have been raised (30,32). In the absence of the definitive isolation, characterization, and identification of key reaction intermediates, an unequivocal NOS mechanism cannot be advanced and therefore several alternative mechanisms have been proposed for each catalytic step.…”

“…Structural Model of Interaction between Fe II -CO and NOS Substrate-The crystallographic structures of NOS-hemeFe II -CO (39) and ferrous heme-nitric oxide complex (39,83), in the presence of L-Arg and NOHA, suggest the existence of an H-bond between the L-Arg-guanidinium and the distal ligand (39,83). Additionally, in the presence of L-Arg, all crystallographic structures reveal the presence of a structural water molecule involved in H-bonding interactions with both the ligand (CO or NO) and the guanidinium moiety of L-Arg (Fig.…”

“…The stronger H-bond between the water and the guanidinium (83) could displace and/or reorient the water molecule and disrupt its H-bond with the CO resulting in an H-bonding situation similar to that of NOS with bound NOHA (see above and Ref. 83). The ensuing change in the distal H-bond network, where the net H-bond interaction on the CO ligand is weaker, gives rise to conformation 2 (Scheme 3B).…”

The Proximal Hydrogen Bond Network Modulates Bacillus subtilis Nitric-oxide Synthase Electronic and Structural Properties

“…The resonance Raman fingerprints of numerous saNOS (39 -41) and bsNOS (42,43) complexes reveal no significant differences in the heme distal environment of bacNOSs and mNOSs (44 -46). Some subtle differences have been observed between the x-ray structures of Fe II NO complexes of bsNOS (47) and neuronal NOS (nNOS) (48,49) in the presence of NOHA and L-Arg, but they should not significantly affect the proton transfer processes. In fact, the variability of the heme distal structure is greater within mNOSs than between bacNOSs and mNOSs.…”

Nitric-oxide Synthase Forms N-NO-pterin and S-NO-Cys

“…However, NO could react with the pterin radical if the process becomes uncoupled or at high NO concentrations, as may occur during inflammatory responses. Also, NOS enzymes have been shown to sequester NO at non-heme sites near the active site (57,58), which would then be available for reaction with a subsequent pterin radical. Moreover, Schmidt and co-workers (59) showed that NOS catalysis leads to H 4 B loss or modification that is distinct from oxidation and dependent on a NOS-derived reactive nitrogen species.…”