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.

This article is dedicated to the memory of Professor A.E. Shilov, the founder of platinum-alkane chemistry. Abstract: A series of Pt(IV) carbene complexes of the form [(Tp′)Pt(=C(X)(Y))(Me) 2 ] + (Tp′ = hydridotris(3,5-dimethylpyrazolyl)borate) was synthesized. Methylating a series of Ptcarboxamido precursors allowed isolation of [(Tp′)Pt(=C(OMe)NHR)Me 2 ][OTf] (R = Et, n Pr, i Pr, Bn). The solid-state structures of the Pt-carbene complexes reveal resonance-stabilized cationic carbene complexes. The method for generating these rare Pt(IV) carbenes was extended to synthesize a methoxy stabilized Pt carbene complex, [Tp′Pt(=C(OCH 3 )CH 3 )Me 2 ][OTf], from a Pt-acyl precursor. Preparation of an alkylidene analog proved more challenging, but low temperature protonation (-80 o C) of a Pt-vinyl complex, (Tp′)Pt(CH=CH 2 )(Me) 2 , generated a small amount of an ethylidene Pt complex as monitored by NMR. Low temperature NMR indicated the presence of the Pt=CH(CH 3 ) unit in [(Tp′)Pt(=CH(CH 3 )Me 2 ][B(Ar F ) 4 ]. The 13 C labeling studies revealed the carbene carbon signal at 500 ppm, a dramatic downfield chemical shift. Warming the reaction to -40 o C results in isomerization accompanied by protonation of a Tp′ nitrogen and reductive elimination to form a new C-C bond in a Pt(II) propylene adduct, [κ 2 (Tp′ (NH) )Pt(η 2 CH 2 =CHCH 3 )Me][B(Ar F ) 4 ]. Abstract GraphicIntroduction:

Scorpionate ligands provide the benefit of hemilability while minimizingcomplete dissociation of the ligand. Previous investigations into Tp′PtL n X m complexes[Tp′= hydridotris(3,5-dimethylpyrazolyl)borate]revealed the importance of hemilabilityas the Tp′ligand facilitates Pt(II/IV) interconversions. Here we discuss the synthesis and metalation of a series of asymmetric scorpionate ligands bearing two pyrazolyl rings and one triazolyl ring. In addition to utilizing triazole donor arms with differing substituents, we also compareoctahedral structures of Pt(IV) complexes with P=O and C-H and B-H caps at the pole of the facial tridentate umbrella.Oxidation from Pt(II) to Pt(IV) with electrophilic reagents, simple acids and acid chlorides, leads to isomers in some cases, and the binding properties of the various donor arms dictatethe stereochemistry of the products. Investigations into the reactivity of heteroscorpionate tridentate ligandsbound to platinum(II)led to C-Cl activation reactions with methylene chloride and 1,2dichloroethane. Isolation of a dinuclear platinum complex bridged by an ethylene unit produced

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.