The role of nitric oxide (NO) in the host response to infection and in cellular signaling is well established. Enzymatic synthesis of NO is catalyzed by the nitric oxide synthases (NOSs), which convert Arg into NO and citrulline using co-substrates O2 and NADPH. Mammalian NOS contains a flavin reductase domain (FAD and FMN) and a catalytic heme oxygenase domain (P450-type heme and tetrahydrobiopterin). Bacterial NOSs, while much less studied, were previously identified as only containing the heme oxygenase domain of the more complex mammalian NOSs. We report here on the characterization of a NOS from Sorangium cellulosum (both fulllength, scNOS, and oxygenase domain, scNOSox). scNOS contains a catalytic, oxygenase domain similar to those found in the mammalian NOS and in other bacteria. Unlike the other bacterial NOSs reported to date, however, this protein contains a fused reductase domain. The scNOS reductase domain is unique for the entire NOS family because it utilizes a 2Fe2S cluster for electron transfer. scNOS catalytically produces NO and citrulline in the presence of either tetrahydrobiopterin or tetrahydrofolate. These results establish a bacterial electron transfer pathway used for biological NO synthesis as well as a unique flexibility in using different tetrahydropterin cofactors for this reaction.heme protein ͉ iron-sulfur cluster ͉ reductase ͉ tetrahydrobiopterin ͉ tetrahydrofolate N itric oxide (NO) is recognized as an important signaling molecule as well as a cytotoxin (1-3). A number of diseases are intimately tied to improper function of NO in humans (1-3). Given the importance of NO to human health, a wealth of studies has been performed to address questions regarding NO synthesis and regulation (4-9). Isolation and structural characterization of the proteins responsible for NO synthesis have led to important developments in understanding the molecular processes of NO formation (10-12). Three isoforms of nitric oxide synthase (NOS), iNOS, eNOS, and nNOS, have been characterized in mammals. These enzymes contain an oxygenase domain where the catalysis takes place and a reductase domain that is involved in electron transfer (4, 9). NOS is functional only as a homodimer, and requires the binding of a Ca 2ϩ -calmodulin (CaM) complex for electron transfer between the reductase and oxygenase domains. The reductase domain transfers electrons from NADPH (nicotinamide adenine dinucleotide phosphate) via FAD (flavin adenine dinucleotide) and FMN (flavin mononucleotide) to the P450-type heme in the oxygenase domain (13). NOS contains an additional reduced pterin cofactor in the oxygenase domain [H 4 B, (6R)-tetrahydro-l-biopterin], which is required for NO formation and is involved in electron transfer processes during catalysis at the heme (14-16).NOS catalyzes the conversion of Arg into NO and citrulline using O 2 and NADPH involving two catalytic steps. In the first reaction, Arg is oxidized to N G -hydroxy-arginine (NHA). Nitric oxide is generated in the second half of the cycle, with the conversion...

X-ray absorption spectroscopy at the L-edge of 3d transition metals provides unique information on the local metal charge and spin states by directly probing 3d-derived molecular orbitals through 2p-3d transitions. However, this soft x-ray technique has been rarely used at synchrotron facilities for mechanistic studies of metalloenzymes due to the difficulties of x-ray-induced sample damage and strong background signals from light elements that can dominate the low metal signal. Here, we combine femtosecond soft x-ray pulses from a free-electron laser with a novel x-ray fluorescence-yield spectrometer to overcome these difficulties. We present L-edge absorption spectra of inorganic high-valent Mn complexes (Mn ∼ 6–15 mmol/l) with no visible effects of radiation damage. We also present the first L-edge absorption spectra of the oxygen evolving complex (Mn4CaO5) in Photosystem II (Mn < 1 mmol/l) at room temperature, measured under similar conditions. Our approach opens new ways to study metalloenzymes under functional conditions.

A concise asymmetric synthesis of (-)-halenaquinone is described. The synthesis features a diastereoselective Heck cyclization to set a quaternary center as well as a novel intramolecular inverse-electron-demand Diels-Alder reaction involving a vinyl quinone. The synthesis is highly convergent and features a minimal amount of protecting group manipulations.

The modulation of the reactivity of metal oxo species by redox inactive metals has attracted much interest due to the observation of redox inactive metal effects on processes involving electron transfer both in nature (the oxygen evolving complex of Photosystem II) and in heterogeneous catalysis (mixed-metal oxides). Studies of small molecule models of these systems have revealed numerous instances of effects of redox inactive metals on electron and group transfer reactivity. However, the heterometallic species directly involved in these transformations have rarely been structurally characterized and are often generated in situ. We have previously reported the preparation and structural characterization of multiple series of heterometallic clusters based on Mn3 and Fe3 cores and described the effects of Lewis acidity of the heterometal incorporated in these complexes on cluster reduction potential. To determine the effects of Lewis acidity of redox inactive metals on group transfer reactivity in structurally well-defined complexes, we studied [Mn3MO4], [Mn3MO(OH)], and [Fe3MO(OH)] clusters in oxygen atom transfer (OAT) reactions with phosphine substrates. The qualitative rate of OAT correlates with the Lewis acidity of the redox inactive metal, confirming that Lewis acidic metal centers can affect the chemical reactivity of metal oxo species by modulating cluster electronics.

Well-defined mixed–metal [CoMn3O4] and [NiMn3O4] cubane complexes were synthesized and used as precursors for heterogeneous oxygen evolution reaction (OER) electrocatalysts. The discrete clusters were dropcasted onto glassy carbon (GC) and indium-tin oxide (ITO) electrodes, and the OER activities of the resulting films were evaluated. The catalytic surfaces were analysed by various techniques to gain insight into the structure-function relationships of the electrocatalysts’ heterometallic composition. Depending on preparation conditions, the Co-Mn-oxide was found to change metal composition during catalysis, while the Ni-Mn-oxides maintained the NiMn3 ratio. XAS studies provided structural insights indicating that the electrocatalysts are different from the molecular precursors, but that the original NiMn3O4 cubane-like geometry was maintained in the absence of thermal treatment (2-Ni). In contrast, the thermally generated 3-Ni develops an oxide-like extended structure. Both 2-Ni and 3-Ni show structural changes upon electrolysis, but they do not convert to the same material. The observed structural motifs in these heterogeneous electrocatalysts are reminiscent of the biological Oxygen Evolving Complex in Photosystem II, including the MMn3O4 cubane moiety. The reported studies demonstrate the use of discrete heterometallic-oxide clusters as precursors for heterogeneous water oxidation catalysts of novel composition and the distinct behavior of two sets of mixed-metal oxides.