Nitrate assimilation in autotrophs provides most of the reduced nitrogen on earth. In eukaryotes, reduction of nitrate to nitrite is catalyzed by the molybdenum-containing NAD(P)H:nitrate reductase (NR; EC 1.7.1.1-3). In addition to the molybdenum center, NR contains iron-heme and flavin adenine dinucleotide as redox cofactors involved in an internal electron transport chain from NAD(P)H to nitrate. Recombinant, catalytically active Pichia angusta nitrate-reducing, molybdenum-containing fragment (NR-Mo) was expressed in P. pastoris and purified. Crystal structures for NR-Mo were determined at 1.7 and 2.6 Å. These structures revealed a unique slot for binding nitrate in the active site and identified key Arg and Trp residues potentially involved in nitrate binding. Dimeric NR-Mo is similar in overall structure to sulfite oxidases, with significant differences in the active site. Sulfate bound in the active site caused conformational changes, as compared with the unbound enzyme. Four ordered water molecules located in close proximity to Mo define a nitrate binding site, a penta-coordinated reaction intermediate, and product release. Because yeast NAD(P)H:NR is representative of the family of eukaryotic NR, we propose a general mechanism for nitrate reduction catalysis.

The circadian clock is a timekeeping mechanism that enables anticipation of daily environmental changes. In the plant Arabidopsis thaliana, the circadian system is a multiloop series of interlocked transcription‐translation feedbacks. Several genes have been arranged in these oscillation loops, but the position of the core‐clock gene ELF4 in this network was previously undetermined. ELF4 lacks sequence similarity to known domains, and functional homologs have not yet been identified. Here we show that ELF4 is functionally conserved within a subclade of related sequences, and forms an alpha‐helical homodimer with a likely electrostatic interface that could be structurally modeled. We support this hypothesis by expression analysis of new elf4 hypomorphic alleles. These weak mutants were found to have expression level phenotypes of both morning and evening clock genes, implicating multiple entry points of ELF4 within the multiloop network. This could be mathematically modeled. Furthermore, morning‐expression...

Nitrate reductase (NR) is important for higher land plants, as it catalyzes the rate-limiting step in the nitrate assimilation pathway, the two-electron reduction of nitrate to nitrite. Furthermore, it is considered to be a major enzymatic source of the important signaling molecule nitric oxide (NO), that is produced in a one-electron reduction of nitrite. Like many other plants, the model plant Arabidopsis thaliana expresses two isoforms of NR (NIA1 and NIA2). Up to now, only NIA2 has been the focus of detailed biochemical studies, while NIA1 awaits biochemical characterization. In this study, we have expressed and purified functional fragments of NIA1 and subjected them to various biochemical assays for comparison with the corresponding NIA2-fragments. We analyzed the kinetic parameters in multiple steady-state assays using nitrate or nitrite as substrate and measured either substrate consumption (nitrate or nitrite) or product formation (NO). Our results show that NIA1 is the more efficient nitrite reductase while NIA2 exhibits higher nitrate reductase activity, which supports the hypothesis that the isoforms have special functions in the plant. Furthermore, we successfully restored the physiological electron transfer pathway of NR using reduced nicotinamide adenine dinucleotide (NADH) and nitrate or nitrite as substrates by mixing the N-and C-terminal fragments of NR, thus, opening up new possibilities to study NR activity, regulation and structure.

Aims: Recent studies suggest that the molybdenum enzymes xanthine oxidase, aldehyde oxidase, and mARC exhibit nitrite reductase activity at low oxygen pressures. However, inhibition studies of xanthine oxidase in humans have failed to block nitrite-dependent changes in blood flow, leading to continued exploration for other candidate nitrite reductases. Another physiologically important molybdenum enzyme—sulfite oxidase (SO)—has not been extensively studied. Results: Using gas-phase nitric oxide (NO) detection and physiological concentrations of nitrite, SO functions as nitrite reductase in the presence of a one-electron donor, exhibiting redox coupling of substrate oxidation and nitrite reduction to form NO. With sulfite, the physiological substrate, SO only facilitates one turnover of nitrite reduction. Studies with recombinant heme and molybdenum domains of SO indicate that nitrite reduction occurs at the molybdenum center via coupled oxidation of Mo(IV) to Mo(V). Reaction rates of nitrite to NO decreased in the presence of a functional heme domain, mediated by steric and redox effects of this domain. Using knockdown of all molybdopterin enzymes and SO in fibroblasts isolated from patients with genetic deficiencies of molybdenum cofactor and SO, respectively, SO was found to significantly contribute to hypoxic nitrite signaling as demonstrated by activation of the canonical NO-sGC-cGMP pathway. Innovation: Nitrite binds to and is reduced at the molybdenum site of mammalian SO, which may be allosterically regulated by heme and molybdenum domain interactions, and contributes to the mammalian nitrate-nitrite-NO signaling pathway in human fibroblasts. Conclusion: SO is a putative mammalian nitrite reductase, catalyzing nitrite reduction at the Mo(IV) center. Antioxid. Redox Signal. 23, 283–294.

The molybdenum cofactor (Moco) forms the catalytic site in all eukaryotic molybdenum enzymes and is synthesized by a multistep biosynthetic pathway. The mechanism of transfer, storage, and insertion of Moco into the appropriate apo-enzyme is poorly understood. In Chlamydomonas reinhardtii, a Moco carrier protein (MCP) has been identified and characterized recently. Here we show biochemical evidence that MCP binds Moco as well as the tungstate-substituted form of the cofactor (Wco) with high affinity, whereas molybdopterin, the ultimate cofactor precursor, is not bound. This binding selectivity points to a specific metal-mediated interaction with MCP, which protects Moco and Wco from oxidation with t_½ of 24 and 96 h, respectively. UV-visible spectroscopy showed defined absorption bands at 393, 470, and 570 nm pointing to ene-diothiolate and protein side-chain charge transfer bonds with molybdenum. We have determined the crystal structure of MCP at 1.6Å resolution using seleno-methionated and native protein. The monomer constitutes a Rossmann fold with two homodimers forming a symmetrical tetramer in solution. Based on conserved surface residues, charge distribution, shape, in silico docking studies, structural comparisons, and identification of an anionbinding site, a prominent surface depression was proposed as a Moco-binding site, which was confirmed by structure-guided mutagenesis coupled to substrate binding studies.