Kinetic consequences of the endogenous ligand to molybdenum in the DMSO reductase family: a case study with periplasmic nitrate reductase.

The molybdopterin enzyme family catalyzes a variety of substrates and plays a critical role in the cycling of carbon, nitrogen, arsenic, and selenium. The dimethyl sulfoxide reductase (DMSOR) subfamily is the most diverse family of molybdopterin enzymes and the members of this family catalyze a myriad of reactions that are important in microbial life processes. Enzymes in the DMSOR family can transform multiple substrates; however, quantitative information about the substrate preference is sparse, and, more importantly, the reasons for the substrate selectivity are not clear. Molybdenum coordination has long been proposed to impact the catalytic activity of the enzyme. Specifically, the molybdenum-coordinating residue may tune substrate preference. As such, molybdopterin enzyme periplasmic nitrate reductase (Nap) is utilized as a vehicle to understand the substrate preference and delineate the kinetic underpinning of the differences imposed by exchanging the molybdenum ligands. To this end, NapA from Campylobacter jejuni has been heterologously overexpressed, and a series of variants, where the molybdenum coordinating cysteine has been replaced with another amino acid, has been produced. The kinetic properties of these variants are discussed and compared with those of the native enzyme, providing quantitative information to understand the function of the molybdenum-coordinating residue.

Molecular cloning, expression and biochemical characterization of periplasmic nitrate reductase from Campylobacter jejuni.

Campylobacter jejuni, a human gastrointestinal pathogen, uses nitrate for growth under microaerophilic conditions using periplasmic nitrate reductase (Nap). The catalytic subunit, NapA, contains two prosthetic groups, an iron sulfur cluster and a molybdenum cofactor. Here we describe the cloning, expression, purification, and Michaelis-Menten kinetics (kcat of 5.91 ± 0.18 s-1 and a KM (nitrate) of 3.40 ± 0.44 µM) in solution using methyl viologen as an electron donor. The data suggest that the high affinity of NapA for nitrate could support growth of C. jejuni on nitrate in the gastrointestinal tract. Site-directed mutagenesis was used and the codon for the molybdenum coordinating cysteine residue has been exchanged for serine. The resulting variant NapA is 4-fold less active than the native enzyme confirming the importance of this residue. The properties of the C. jejuni enzyme reported here represent the first isolation and characterization of an epsilonproteobacterial NapA. Therefore, the fundamental knowledge of Nap has been expanded.

Upon further analysis, neither cytochrome c554 from Nitrosomonas europaea nor its F156A variant display NO reductase activity, though both proteins bind nitric oxide reversibly.

A re-investigation of the interaction with NO of the small tetraheme protein cytochrome c554 (C554) from Nitrosomonas europaea has shown that the 5-coordinate heme II of the two- or four-electron-reduced protein will nitrosylate reversibly. The process is first order in C554, first order in NO, and second-order overall. The rate constant for NO binding to the heme is 3000 ± 140 M-1s-1, while that for dissociation is 0.034 ± 0.009 s-1; the degree of protein reduction does not appear to significantly influence the nitrosylation rate. In contrast to a previous report (Upadhyay AK, et al. J Am Chem Soc 128:4330, 2006), this study found no evidence of C554-catalyzed NO reduction, either with [Formula: see text] or with [Formula: see text] Some sub-stoichiometric oxidation of the lowest potential heme IV was detected when [Formula: see text] was exposed to an excess of NO, but this is believed to arise from partial intramolecular electron transfer that generates {Fe(NO)}8 at heme II. The vacant heme II coordination site of C554 is crowded by three non-bonding hydrophobic amino acids. After replacing one of these (Phe156) with the smaller alanine, the nitrosylation rate for F156A2- and F156A4- was about 400× faster than for the wild type, though the rate of the reverse denitrosylation process was almost unchanged. Unlike in the wild-type C554, the 6-coordinate low-spin hemes of F156A4- oxidized over the course of several minutes after exposure to NO. Concomitant formation of N2O could explain this heme oxidation, though alternative explanations are equally plausible given the available data.

Interaction of nitric oxide with catalase: structural and kinetic analysis.

We present the structures of bovine catalase in its native form and complexed with ammonia and nitric oxide, obtained by X-ray crystallography. Using the NO generator 1-(N,N-diethylamino)diazen-1-ium-1,2-diolate, we were able to generate sufficiently high NO concentrations within the catalase crystals that substantial occupation was observed despite a high dissociation rate. Nitric oxide seems to be slightly bent from the heme normal that may indicate some iron(II) character in the formally ferric catalase. Microspectrophotometric investigations inline with the synchrotron X-ray beam reveal photoreduction of the central heme iron. In the cases of the native and ammonia-complexed catalase, reduction is accompanied by a relaxation phase. This is likely not the case for the catalase NO complex. The kinetics of binding of NO to catalase were investigated using NO photolyzed from N,N'-bis(carboxymethyl)-N,N'-dinitroso-p-phenylenediamine using an assay that combines catalase with myoglobin binding kinetics. The off rate is 1.5 s(-1). Implications for catalase function are discussed.