Gating NO Release from Nitric Oxide Synthase

We have investigated the kinetics of NO escape from Geobacillus stearothermophilus nitric oxide synthase (gsNOS). Previous work indicated that NO release was gated at position 223 in mammalian enzymes; our kinetics experiments include mutants at that position along with measurements on the wild type enzyme. Employing stopped-flow UV–vis methods, reactions were triggered by mixing a reduced enzyme/N-hydroxy-l-arginine complex with an aerated buffer solution. NO release kinetics were obtained for wt NOS and three mutants (H134S, I223V, H134S/I223V). We have confirmed that wt gsNOS has the lowest NO release rate of known NOS enzymes, whether bacterial or mammalian. We also have found that steric clashes at positions 223 and 134 hinder NO escape, as judged by enhanced rates in the single mutants. The empirical rate of NO release from the gsNOS double mutant (H134/I223V) is nearly as rapid as that of the fastest mammalian enzymes, demonstrating that both positions 223 and 134 function as gates for escape of the product diatomic molecule

Nanosecond photoreduction of inducible nitric oxide synthase by a Ru-diimine electron tunneling wire bound distant from the active site

A Ru-diimine wire, [(4,4′,5,5′-tetramethylbipyridine)_2Ru(F_9bp)]^(2+) (tmRu-F_9bp, where F_9bp is 4-methyl-4′-methylperfluorobiphenylbipyridine), binds tightly to the oxidase domain of inducible nitric oxide synthase (iNOSoxy). The binding of tmRu-F_9bp is independent of tetrahydrobiopterin, arginine, and imidazole, indicating that the wire resides on the surface of the enzyme, distant from the active-site heme. Photoreduction of an imidazole-bound active-site heme iron in the enzyme-wire conjugate (k_(ET) = 2(1) × 10^7 s^(−1)) is fully seven orders of magnitude faster than the in vivo process

Probing the heme-thiolate oxygenase domain of inducible nitric oxide synthase with Ru(II) and Re(I) electron tunneling wires

Nitric oxide synthase (NOS) catalyzes the production of nitric oxide from L-arginine and dioxygen at a thiolate-ligated heme active site. Although many of the reaction intermediates are as yet unidentified, it is well established that the catalytic cycle begins with substrate binding and rate-limiting electron transfer to the heme. Here, we show that Ru(II)-diimine and Re(I)-diimine electron tunneling wires trigger nanosecond photoreduction of the active-site heme in the enzyme. Very rapid generation of a reduced thiolate-ligated heme opens the way for direct observation of short-lived intermediates in the NOS reaction cycle

Kinetics of CO recombination to the heme in Geobacillus stearothermophilus nitric oxide synthase

We report the kinetics of CO rebinding to the heme in His134Ser, Ile223Val and His134Ser/Ile223Ser mutants of Geobacillus stearothermophilus nitric oxide synthase (gsNOS). The amplitudes of the two observed kinetics phases, which are insensitive to CO concentration, depend on enzyme concentration. We suggest that two forms of gsNOS are in equilibrium under the conditions employed (6.1–27 μM gsNOS with 20 or 100% CO atmosphere). The kinetics of CO rebinding to the heme do not depend on the identity of the NO-gate residues at positions 134 and 223

Tuning Nitric Oxide Synthase: Investigating the Thiolate "Push" and No Release

All heme thiolate enzymes have conserved hydrogen bonding networks surrounding the axial thiolate ligand. In order to understand the role of this proximal hydrogen bonding network in nitric oxide synthases (NOS), three mutants of the NOS enzyme from Geobacillus stearothermophilus were expressed and characterized. The wild type enzyme has a tryptophan residue at position 70 that π-stacks with the porphyrin ring and donates a long hydrogen-bonding interaction to the thiolate ligand of the heme iron. The native Trp was replaced with His, Phe, and Tyr. These three residues were selected to investigate the two effects of the Trp, H-bonding and Pi-stacking. Several different spectroscopic techniques were used to investigate the stability and properties of these mutant enzymes. The identity of each mutant was confirmed by mass spectrometry. Both UV-visible absorption and circular dichroism spectroscopies were used to assess the stability of the new proteins. It was shown using binding assays, generation of the ferrous-CO species, and redox titrations that the σ-donating abilities of the thiolate are increased after removal of the hydrogen bonding group in the Trp. Finally, electron paramagnetic resonance spectroscopy and Evans method nuclear magnetic resonance spectroscopy were used to characterize the spin state of the iron center in each mutant, reflecting the increased σ-donating capabilities of the thiolate upon removal of the hydrogen bonding group. The reduction potential of wild type and W70H were determined by chemical titration to be -362 and -339 mV vs. NHE, respectively. This is the first report of the reduction potential of any bacterial nitric oxide synthase. The reactivity of each the wild type enzyme and the three new mutants was tested using stopped-flow mixing coupled with UV-visible absorption spectroscopy and the Griess Assay. Autoxidation rates measured by stopped-flow suggest that the Tyr and Phe mutants do indeed have significantly more negative reduction potentials, but that the His mutant is particularly slow to oxidize. The Griess Assays showed that all four enzymes produce nitrite in solution, when provided with substrate, cofactor and hydrogen peroxide (as a source of reducing equivalents). In single turnover experiments, however, only three of the four enzymes showed evidence of ferric-NO production. The His mutant showed no intermediate absorbance near 440 nm (which would be indicative of ferric-NO formation), suggesting that it releases NO- rather than the radical species NO∙. The role of this hydrogen bond is concluded to be an electronic one, rather than playing any part in positioning the heme. It prevents formation of the inactive P420 species, and tunes the reduction potential to one high enough to be reduced by a reductase but low enough to still deliver an electron to the redox active cofactor, tetrahydrobiopterin, at the end of catalysis. The rate at which NO is released by each NOS enzyme varies greatly among isoforms and species, over nearly two orders of magnitude. One residue (an isoleucine located above the heme in bacterial enzymes) involved in the gating of NO release has been previously identified by Stuehr. However, this single residue does not account for the entirety of the differences among the forms of NOS. Another residue, a histidine at position 134 in NOS from Geobacillus stearothermophilus (gsNOS), was hypothesized to also participate in gating NO release based on an observed correlation between rates of NO release and the bulk of side chains at this position. Each single point mutation, H134S and I223V, and the double mutant were expressed in gsNOS and their reactivity toward the diatomic molecules CO and NO were studied. CO rebinding was investigated using laser flash photolysis and NO release using stopped flow UV-visible spectroscopy. The presence of both monomer and dimer was observed in solution, and position 134 was shown to be another key residue in gating NO release. Wild type gsNOS contains both the bulkier Ile223 and His134 and has the slowest measured NO release (0.039 s-1) of all NOS enzymes. A new, more accurate kinetics model for turnover is proposed. Each single mutation increased NO release substantially, while the double mutant has a rate constant of 1.0 s-1, nearly as fast as mammalian iNOS at 2.3 s-1, identifying position 134 as another important factor determining rate constants for NO release.</p

Determinants of Ligand Affinity and Heme Reactivity in H-NOX Domains

O_2 balks at extra bulk: The introduction of distal-pocket bulk into the Thermoanaerobacter tengcongensis H-NOX (heme nitric oxide/oxygen) domain caused key changes in the protein structure. Rearrangement of the heme pocket resulted in dramatic differences in O_2-binding kinetics and heme reactivity (see picture)