Kinetics of CO Ligation with Nitric-oxide Synthase by Flash Photolysis and Stopped-flow Spectrophotometry

Abstract

Interaction of CO with hemeproteins has physiological importance. This is especially true for nitric-oxide synthases (NOS), heme/flavoenzymes that produce ⅐ NO and citrulline from L-arginine (Arg) and are inhibited by CO in vitro. The kinetics of CO ligation with both neuronal NOS and its heme domain module were determined in the presence and absence of tetrahydrobiopterin and Arg to allow comparison with other hemeproteins. Geminate recombination in the nanosecond time domain is followed by bimolecular association in the millisecond time domain. Complex association kinetics imply considerable heterogeneity but can be approximated with two forms, one fast (2-3 ؋ Nitric-oxide synthase is a hemoprotein that catalyzes conversion of the amino acid L-arginine to citrulline and nitric oxide (1, 2). The NO produced activates another heme enzyme, guanylate cyclase (see Reaction 1), that catalyzes formation of cyclic guanosine monophosphate, a second messenger that mediates numerous biochemical events including vascular smooth muscle relaxation, platelet disaggregation, photoreceptor cell signaling, ion transport in gastrointestinal cells, and myeloid cell differentiation.At least three isoforms of NOS 1 have been purified and characterized. In each, a heme prosthetic group, iron protoporphyrin IX, is attached to the protein by a proximal cysteine. Visible absorption spectroscopy and other physicochemical measurements reveal similarities to cytochromes P450 (3-8).Like that family of proteins, reduced (ferro) NOS forms carboxy derivatives with an absorption maximum at ϳ446 nm (4). Marletta (9) and Stuehr et al. (10) have proposed mechanisms for the oxidation of Arg catalyzed by NOS, based largely upon the similarity of NOS and P450, and Masters (11) has proposed a scheme involving redox interactions with the flavoprotein module of NOS. A key observation was that enzyme activity is inhibited by CO (4, 6, 9) as well as by NO (12), the physiological significance of which remains uncertain. Enzyme inhibition in vitro required large concentrations of either CO or NO. If those reagents play a role in vivo, the mechanism must involve locally enhanced concentrations. For NO, fast turnover and slow diffusion away from the site of production in unstirred cytoplasm might possibly lead to large concentrations locally and, consequently, to autoregulation of NO production. Recent evidence suggests a role for CO produced by heme oxygenase as a messenger molecule in the same cell types as nNOS (13-15). Could the activity of heme oxygenase produce high concentrations? The CO concentration needed depends upon the equilibrium constant for CO binding. Unfortunately, equilibrium constants for binding CO and NO are not known for NOS. Matsuoka et al. (16) reported CO combination rate constants for neuronal NOS but not CO dissociation data, which could have been combined with the combination rate constant to calculate an equilibrium constant. In hemeproteins, CO dissociation rate constants vary from 6.5 s Ϫ1 in P450 to 7.2 ϫ 10 Ϫ5 s Ϫ1 in hors...

Heme Distortion Modulated by Ligand-Protein Interactions in Inducible Nitric-oxide Synthase

“…Based on the crystal structures of the oxygenase domain of iNOS (iNOS oxy ), Crane et al (8) concluded that H4B binding resulted in major conformational changes to the protein that are critical for the promotion of subunit assembly into a dimer, the active form of NOS, and the formation of the reductase docking site required for the electron transfer. In addition, biochemical studies of various isoforms of NOS, showed that H4B binding introduces significant changes in protein stability, monomer/dimer equilibrium, proteolytic susceptibility, heme-ligand binding, and substrate binding properties (21)(22)(23)(24)(25)(26). In contrast, based on the crystal structure of the oxygenase domain of eNOS (eNOS oxy ), Raman et al (11) reported that H4B binding does not produce any conformational changes in the protein, and more importantly the dimeric assembly is retained in the absence of H4B.…”

Kinetics of CO and NO Ligation with the Cys331 → Ala Mutant of Neuronal Nitric-oxide Synthase

“…2 shows the fraction of the slow phase in two circumstances for C331A. It also includes, for comparison, data on the WT holoenzyme and data for a heme-binding domain (residues 1-714), both of which were reported previously (14). The fraction of the slow phase is at its maximum when both L-Arg and BH 4 are bound.…”

“…As with the WT holoenzyme and heme domain (14), flash photolysis of the CO-ligated C331A mutant displays, in addition to bimolecular association, nanosecond transient absorbance changes that are plausibly attributed to geminate recombination. In all cases, these nanosecond transients are larger whenever the fast phase is larger.…”

“…1. This was discussed at some length for WT (14). Once the heterogeneity is noted, it is sufficient for most purposes to consider an effective mean rate.…”

“…Flash photolysis was carried out at 22°C using procedures described previously (14,15 flow measurements were carried out at 20°C following protocols described earlier (16). Carbon monoxide dissociation rates were determined by replacing CO with NO (17).…”