O2 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 O2‐binding kinetics and heme reactivity (see picture).

Scheuer Sauerstoff: Das Einführen von sterischer Belastung in der entfernten Tasche der Thermoanaerobacter‐tengcongensis‐H‐NOX‐Domäne (H‐NOX: Häm‐Stickstoffmonoxid/Sauerstoff) verändert die Proteinstruktur erheblich, was drastische Unterschiede in der O2‐Bindekinetik und der Häm‐Reaktivität zur Folge hat (siehe Bild).

We have investigated the kinetics of NO escape from Geobacillus stearothermophilus nitric oxide synthase (gsNOS). Previous work has 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 reduced enzyme/N-hydroxy-L-arginine complex with 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.

A Ru-diimine wire, [(4,4’,5,5’-tetramethylbipyridine)2Ru(F9bp)]2+ (tmRu-F9bp, where F9bp is 4-methyl-4’-methylperfluorobiphenylbipyridine), binds tightly to the oxidase domain of inducible nitric oxide synthase (iNOSoxy). The binding of tmRu-F9bp 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 (kET = 2(1) × 107 s-1) is fully seven orders of magnitude faster than the in vivo process.

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.