No abstract

High resolution x-ray crystallographic structures of nitrite reductase from Achromobacter cycloclastes, undertaken in order to understand the pH optimum of the reaction with nitrite, show that at pH 5.0, 5.4, 6.0, 6.2, and 6.8, no significant changes occur, other than in the occupancy of the type II copper at the active site. An extensive network of hydrogen bonds, both within and between subunits of the trimer, maintains the rigidity of the protein structure. A water occupies a site approximately 1.5 A from the site of the type II copper in the structure of the type II copper-depleted structure (at pH 5.4), again with no other significant changes in structure. In nitrite-soaked crystals, nitrite binds via its oxygens to the type II copper and replaces the water normally bound to the type II copper. The active-site cavity of the protein is distinctly hydrophobic on one side and hydrophilic on the other, providing a possible path for diffusion of the product NO. Asp-98 exhibits thermal parameter values higher than its surroundings, suggesting a role in shuttling the two protons necessary for the overall reaction. The strong structural homology with cupredoxins is described.

The three-dimensional crystal structure of the copper-containing nitrite reductase (NIR) from Achromobacter cycloclastes has been determined to 2.3 angstrom (A) resolution by isomorphous replacement. The monomer has two Greek key beta-barrel domains similar to that of plastocyanin and contains two copper sites. The enzyme is a trimer both in the crystal and in solution. The two copper atoms in the monomer comprise one type I copper site (Cu-I; two His, one Cys, and one Met ligands) and one putative type II copper site (Cu-II; three His and one solvent ligands). Although ligated by adjacent amino acids Cu-I and Cu-II are approximately 12.5 A apart. Cu-II is bound with nearly perfect tetrahedral geometry by residues not within a single monomer, but from each of two monomers of the trimer. The Cu-II site is at the bottom of a 12 A deep solvent channel and is the site to which the substrate (NO2-) binds, as evidenced by difference density maps of substrate-soaked and native crystals.

No abstract

The structures of oxidized, reduced, nitrite-soaked oxidized and nitrite-soaked reduced nitrite reductase from Alcaligenes faecalis have been determined at 1.8 -2.0 Å resolution using data collected at ؊160°C. The active site at cryogenic temperature, as at room temperature, contains a tetrahedral type II copper site liganded by three histidines and a water molecule. The solvent site is empty when crystals are reduced with ascorbate. A fully occupied oxygen-coordinate nitrite occupies the solvent site in crystals soaked in nitrite. Ascorbate-reduced crystals soaked in a glycerol-methanol solution and nitrite at ؊40°C remain colorless at ؊160°C but turn amber-brown when warmed, suggesting that NO is released. Nitrite is found at one-half occupancy. Five new solvent sites in the oxidized nitrite bound form exhibit defined but different occupancies in the other three forms. These results support a previously proposed mechanism by which nitrite is bound primarily by a single oxygen atom that is protonable, and after reduction and cleavage of that N-O bond, NO is released leaving the oxygen atom bound to the Cu site as hydroxide or water.

Results from refinement of the crystal structures of P. aerogenes ferredoxin and C pasteurianum rubredoxin determined by x-ray diffraction show that there are 15-18 NHOS bonds in the former and six in the latter with lengths in the range 3.1-3.9 A. Earlier tritium exchange experiments are consistent with the presence of these hydrogen onds in the ferredoxin structure and show that more peptide hydrogen atoms are available for exchange in apoferredoxin than in intact ferredoxin. Four types of NH-S bonds are observed and two of these are geometrically similar to the two.types of 310 NH-O bonds. The existence of more NH-S bonds in ferredoxin than in high potential iron protein suggests why the -2 form of the Fe4S4 cluster is preferred in ferredoxin over the -1 form found in high potential iron protein. From comparison of Cys-X-Y-Cys sequences in rubredoxin, terredoxin, and high potential iron protein we suggest that two Cys-X-Y-Cys-Z sequences, where Z may have conformation angles similar to glycine, are required to make a oneiron cluster, no more than one Cys-X-Y-Cys-Z-Gly sequence is required to form a Fe2S2 ferredoxin, and a Cys-X-Y-Cys-Gly sequence where Y has a conformation such that the cysteines bond to different iron atoms is necessary to form the tetrameric cluster.The properties of iron-sulfur proteins raise an intriguing question: How does the protein structure contribute to the various observed forms of iron-sulfur centers? Iron-sulfur proteins include: (i) rubredoxin (Rb) with one Fe, no inorganic sulfur, and four cysteines; (ii) plant-type ferredoxins with two irons, two inorganic sulfur atoms, and at least four cysteines; and (iii) bacterial-type ferredoxin with one or more tetrameric clusters having four iron atoms, four inorganic sulfur atoms, and four cysteinyl sulfur atoms (1). The latter clusters are found both in Peptococcus aerogenes ferredoxin (Fd) (2) and in high potential iron protein (HiPIP) (3), a bacterial protein with unknown function which is reduced approximately +300 mV, in contrast to ferredoxin, which is reduced at the very low potential of about -400 mV. According to the three-state hypothesis (4), three oxidation states are accessible to the tetrameric cluster, one pair of which is preferred in HiPIP, another pair, of lower potential, in Fd. It has been demonstrated that the cluster in HiPIP can be "superreduced" in the presence of the denaturant dimethyl sulfoxide to the lowest state accessible to Fd (5), while the cluster in ferredoxin can be "superoxidized" in the presence of K3Fe(CN)6 (6). Studies with model compounds for tetrameric clusters have indicated that the -2/-3 reduction step analogous to reduction of ferredoxin operates at a much more negative potential than is observed for the proteins (7). Thus the protein portion of the iron-sulfur proteins has at least two functions that are important for cluster formation and activity: It provides an environment which favors one form of iron-sulfur center over another (i.e., 1 Fe, 2 Fe-2 S, 4 Fe-4 S) and, in the case o...

Nitrite reductase (NIR) from the denitrifying bacterium Alcaligenes faecalis S-6 is a copper-containing enzyme which requires pseudoazurin, a low molecular weight protein containing a single type I copper atom, as a direct electron donor in vivo. Crystallographic analysis shows that NIR is a trimer composed of three identical subunits, each of which contains one atom of type I copper and one atom of type II copper, and that the ligands to the type I and type II copper atoms are the same as those of the Achromobacter cycloclastes NIR. An efficient NIR expression-secretion system in Escherichia coli was constructed and used for site-directed mutagenesis. An NIR mutant with a replacement of the type II copper ligand, His135, by Lys still retained a type II copper site as well as a type I copper atom, but it completely lost nitrite-reducing activity as measured with methyl viologen as an electron donor. On the other hand, another mutant with a replacement of the type I copper ligand, Met150, by Glu contained only a type II copper atom, but it still retained significant nitrite-reducing activity with methyl viologen. When pseudoazurin was used as an electron donor for the reaction, however, Met150Glu failed to catalyze the reduction of nitrite. Kinetic analysis of the electron transfer between NIR and pseudoazurin revealed that the electron-transfer rate between Met150Glu and pseudoazurin was reduced 1000-fold relative to that of wild-type NIR.(ABSTRACT TRUNCATED AT 250 WORDS)

No abstract