In a continuing effort to unravel mechanistic questions associated with metalloenzymes, we are developing methods for rapid delivery of electrons to deeply buried active sites. Herein, we report picosecond reduction of the heme active site of inducible nitric oxide synthase bound to a series of rhenium-diimine electron-tunneling wires, [Re(CO)3LL′] + , where L is 4,7-dimethylphenanthroline and L′ is a perfluorinated biphenyl bridge connecting a rhenium-ligated imidazole or aminopropylimidazole to a distal imidazole (F8bpim (1) and C3-F8bp-im (2)) or F (F9bp (3) and C3-F9bp (4)). All four wires bind tightly (Kd in the micromolar to nanomolar range) to the tetrahydrobiopterin-free oxidase domain of inducible nitric oxide synthase (iNOSoxy). The two fluorine-terminated wires displace water from the active site, and the two imidazoleterminated wires ligate the heme iron. Upon 355-nm excitation of iNOSoxy conjugates with 1 and 2, the active site Fe(III) is reduced to Fe(II) within 300 ps, almost 10 orders of magnitude faster than the naturally occurring reduction.

Ru(II)-and Re(I)-diimine wires bind to the oxygenase domain of inducible nitric oxide synthase (iNOSoxy). In the ruthenium wires, [Ru(L)2L′] 2+ , L′ is a perfluorinated biphenyl bridge connecting 4,4′dimethylbipyridine to a bulky hydrophobic group (adamantane, 1), a heme ligand (imidazole, 2), or F (3). 2 binds in the active site of the murine iNOSoxy truncation mutants ∆65 and ∆114, as demonstrated by a shift in the heme Soret from 422 to 426 nm. 1 and 3 also bind ∆65 and ∆114, as evidenced by biphasic luminescence decay kinetics. However, the heme absorption spectrum is not altered in the presence of 1 or 3, and Ru-wire binding is not affected by the presence of tetrahydrobiopterin or arginine. These data suggest that 1 and 3 may instead bind to the distal side of the enzyme at the hydrophobic surface patch thought to interact with the NOS reductase module. Complexes with properties similar to those of the Rudiimine wires may provide an effective means of NOS inhibition by preventing electron transfer from the reductase module to the oxygenase domain. Rhenium-diimine wires, [Re(CO) 3L1L1′] + , where L1 is 4,7dimethylphenanthroline and L1′ is a perfluorinated biphenyl bridge connecting a rhenium-ligated imidazole to a distal imidazole (F8bp-im) (4) or F (F9bp) (5), also form complexes with ∆114. Binding of 4 shifts the ∆114 heme Soret to 426 nm, demonstrating that the terminal imidazole ligates the heme iron. Steadystate luminescence measurements establish that the 4:∆114 dissociation constant is 100 ( 80 nM. Rewire 5 binds ∆114 with a Kd of 5 ( 2 µM, causing partial displacement of water from the heme iron. Our finding that both 4 and 5 bind in the NOS active site suggests novel designs for NOS inhibitors. Importantly, we have demonstrated the power of time-resolved FET measurements in the characterization of small molecule:protein interactions that otherwise would be difficult to observe.

We report direct electrochemistry of the iNOS heme domain in a DDAB film on the surface of a basal plane graphite electrode. Cyclic voltammetry reveals Fe III/II and Fe II/I couples at −191 and −1049 mV (vs. Ag/AgCl). Added ligands imidazole and carbon monoxide shift the Fe III/II potential by +20 and +62 mV, while the addition of dioxygen results in large catalytic waves at the onset of Fe III reduction. Voltammetry at higher scan rates reveals that the Fe III/II cathodic peak can be resolved into two components, which are attributable to Fe III/II couples of 5-and 6-coordinate hemes. Digital simulation of our experimental data implicates water dissociation from the heme as a gating mechanism for ET in iNOS.The critical role of nitric oxide (NO) in cellular signaling is firmly established. 1 In higher animals, NO is synthesized by nitric oxide synthase (NOS), which converts L-arginine to citrulline and NO with NADPH and O 2 as co-substrates. 2 The enzyme consists of a reductase domain where electrons from NADPH are shuttled through bound flavins FAD and FMN, a calmodulin binding region that controls electron transfer (ET) to the heme, and a heme domain that contains a cysteine-ligated heme and tetrahydrobiopterin (H 4 B). Investigations herein utilize the heme domain of inducible NOS (iNOS), an immune system isoform implicated in several diseases in humans. 3The complexity of NOS and its impact on human health have thrust work on the NOS mechanism into the spotlight. It is well established that the catalytic cycle involves two turnovers of the enzyme. The first turnover converts L-arginine to N-hydroxyarginine: although envisioned as a P450-like hydroxylation, the reaction is dependent on one-electron oxidation of H 4 B. 4 The second turnover is thought to involve a ferric peroxide nucleophile, hbgray@caltech.edu. but H 4 B is also required for this turnover in an as yet undetermined role. Although many details of the NOS mechanism remain to be elucidated, it is certain that ET reactions are key steps in the catalytic cycle. NIH Public AccessDirect electrochemistry of iNOS can be achieved by confining the protein in didodecyldimethylammonium bromide (DDAB) films on the surface of basal plane graphite electrodes (BPG). 5-7 We report reduction potentials for Fe III/II and Fe II/I couples, Fe III/II ET kinetics, catalytic reduction of dioxygen, and evidence for water-free and water-bound forms of the iNOS heme based on scan rate and pH dependence data.DDAB films were formed on BPG (0.07 cm 2 ) by depositing 5 μL of a 10 mM aqueous solution of DDAB on the electrode surface, followed by slow drying overnight. iNOS was incorporated into the film by soaking the coated electrode in a solution of enzyme (~ 20 μM in 50 mM KP i , 50 mM KCl, pH 7 buffer) for 30 minutes. A voltammogram of iNOS in DDAB on BPG is shown in Figure 1. We have assigned E 1 (−191 mV) and E 2 (−1049 mV) to heme Fe III/II and Fe II/I couples, consistent with other studies of heme proteins in DDAB films. 8,9 Notably, a couple similar to E 2 w...