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.

Cyclic nucleotide phosphodiesterases (PDEs) regulate all pathways that use cGMP or cAMP as a second messenger. Five of the 11 PDE families have regulatory segments containing GAF domains, 3 of which are known to bind cGMP. In PDE2 binding of cGMP to the GAF domain causes an activation of the catalytic activity by a mechanism that apparently is shared even in the adenylyl cyclase of Anabaena, an organism separated from mouse by 2 billion years of evolution. The 2.9-Å crystal structure of the mouse PDE2A regulatory segment reported in this paper reveals that the GAF A domain functions as a dimerization locus. The GAF B domain shows a deeply buried cGMP displaying a new cGMP-binding motif and is the first atomic structure of a physiological cGMP receptor with bound cGMP. Moreover, this cGMP site is located well away from the region predicted by previous mutagenesis and structural genomic approaches. C yclic nucleotide phosphodiesterases (PDEs) catalyze the hydrolysis of 3Ј, 5Ј cyclic nucleotides to the inactive 5Ј monophosphates. Five of the 11 PDE families contain regulatory segments consisting of one or two so-called GAF-domain modules (1), which is one of the largest families of small moleculebinding regulatory domains. Among PDEs, cGMP is the only ligand known to bind this domain. The structure of a single GAF domain from a putative protein from yeast (YKG9) has been solved recently (2). However, yeast do not make cGMP, nor does this protein bind cGMP when tested directly (2). cGMP binding to one of two GAF domains (3) in the photoreceptor PDE6 family provides one mechanism for regulating visual signal transduction. cGMP also binds to one or more of the GAF domains of PDE5 (4), the target of the drug, Viagra. The binding and subsequent phosphorylation of an adjacent domain activates the catalytic domain of the enzyme (5). In PDE2A, the catalytic activity is allosterically stimulated by cGMP binding to its GAF domain (6), an event important for several pathways that PDE2A has been shown to regulate (7-12). For example, atrial natriuretic peptide stimulation of cGMP and subsequent activation of PDE2A in the adrenal cortex decreases the secretion of aldosterone and, thereby, mediates much of the effect of this hormone on blood pressure (13). Each PDE2A monomer contains an N-terminal (Ϸ200 residues) domain of unknown function, tandem GAF domains (GAF A and GAF B), and a C-terminal catalytic domain. What seems to be a functionally very similar tandem set of GAF domains is also present in Anabaena adenylyl cyclase. This GAF domain has a preference for cAMP where it functions to confer cAMP activation of cyclase activity (14). Here, we report the 2.9-Å crystal structure of the regulatory segment of murine PDE2A, which reveals the structure of two GAF domains with entirely different functions, dimerization, and binding of cGMP. Amazingly, this binding motif and mechanism has apparently been preserved for over 2 billion years in evolution. Methods and MaterialsCrystallization and Data Collection. Crystals were grown at ...

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.

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.

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