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HD-GYPs make up a subclass of the metal-dependent HD phosphohydrolase superfamily and catalyze conversion of cyclic di(3',5')-guanosine monophosphate (c-di-GMP) to 5'-phosphoguanylyl-(3'→5')-guanosine (pGpG) and GMP. Until now, the only reported crystal structure of an HD-GYP that also exhibits c-di-GMP phosphodiesterase activity contains a His/carboxylate ligated triiron active site. However, other structural and phylogenetic correlations indicate that some HD-GYPs contain dimetal active sites. Here we provide evidence that an HD-GYP c-di-GMP phosphodiesterase, TM0186, from Thermotoga maritima can accommodate both di- and trimetal active sites. We show that an as-isolated iron-containing TM0186 has an oxo/carboxylato-bridged diferric site, and that the reduced (diferrous) form is necessary and sufficient to catalyze conversion of c-di-GMP to pGpG, but that conversion of pGpG to GMP requires more than two metals per active site. Similar c-di-GMP phosphodiesterase activities were obtained with divalent iron or manganese. On the basis of activity correlations with several putative metal ligand residue variants and molecular dynamics simulations, we propose that TM0186 can accommodate both di- and trimetal active sites. Our results also suggest that a Glu residue conserved in a subset of HD-GYPs is required for formation of the trimetal site and can also serve as a labile ligand to the dimetal site. Given the anaerobic growth requirement of T. maritima, we suggest that this HD-GYP can function in vivo with either divalent iron or manganese occupying di- and trimetal sites. [ABSTRACT FROM AUTHOR]

The efficient clearance of microbes by neutrophils requires the concerted action of reactive oxygen species and microbicidal components within leukocyte secretory granules. Rubrerythrin (Rbr) is a nonheme iron protein that protects many air-sensitive bacteria against oxidative stress. Using oxidative burst-knockout (NADPH oxidase-null) mice and an rbr gene knockout bacterial strain, we investigated the interplay between the phagocytic oxidative burst of the host and the oxidative stress response of the anaerobic periodontal pathogen Porphyromonas gingivalis. Rbr ensured the proliferation of P. gingivalis in mice that possessed a fully functional oxidative burst response, but not in NADPH oxidase-null mice. Furthermore, the in vivo protection afforded by Rbr was not associated with the oxidative burst responses of isolated neutrophils in vitro. Although the phagocyte-derived oxidative burst response was largely ineffective against P. gingivalis infection, the corresponding oxidative response to the Rbr-positive microbe contributed to host-induced pathology via potent mobilization and systemic activation of neutrophils. It appeared that Rbr also provided protection against reactive nitrogen species, thereby ensuring the survival of P. gingivalis in the infected host. The presence of the rbr gene in P. gingivalis also led to greater oral bone loss upon infection. Collectively, these results indicate that the host oxidative burst paradoxically enhances the survival of P. gingivalis by exacerbating local and systemic inflammation, thereby contributing to the morbidity and mortality associated with infection. [ABSTRACT FROM AUTHOR]

Several members of a widespread class of bacterial and archaeal metalloflavoproteins, called FprA, likely function as scavenging nitric oxide reductases (S-NORs). However, the only published X-ray crystal structure of an FprA is for a protein characterized as a rubredoxin:dioxygen oxidoreductase (ROO) from Desulfovibrio gigas. Therefore, the crystal structure of Moorella thermoacetica FprA, which has been established to function as an S-NOR, was solved in three different states: as isolated, reduced, and reduced, NO-reacted. As is the case for D. gigas ROO, the M. thermoacetica FprA contains a solvent- bridged non-heme, non-sulfur diiron site with five-coordinate iron centers bridged by an aspartate, and terminal glutamate, aspartate, and histidine ligands. However, the M. thermoacetica FprA diiron site showed four His ligands, two to each iron, in all three states, whereas the D. gigas ROO diiron site was reported to contain only three His ligands, even though the fourth His residue is conserved. The Fel -Fe2 distance within the diiron site of M. thermoacetica FprA remained at 3.2-3.4 Å with little or no movement of the protein ligands in the three different states and with conservation of the two proximal open coordination sites. Molecular modeling indicated that each open coordination site can accommodate an end-on NO. This relatively rigid and symmetrical diiron site structure is consistent with formation of a diferrous dinitrosyl as the committed catalytic intermediate leading to formation of N2O. These results provide new insight into the structural features that fine-tune biological non-heme diiron sites for dioxygen activation vs nitric oxide reduction. [ABSTRACT FROM AUTHOR]

X-ray crystal structures of recombinant Desulfovibrio (D.) vulgaris rubrerythrin (Rbr) have shown a diiron site, whereas the crystal structure of Rbr “as-isolated” from D. vulgaris was reported to contain a mixed Zn,Fe binuclear site. To investigate the possibility that zinc had displaced iron during isolation or crystallization of the “as-isolated” D. vulgaris Rbr, the X-ray crystal structure of recombinant D. vulgaris all-iron Rbr that had been incubated with excess zinc sulfate prior to crystallization, yielding a protein labeled Zn,FeRbr, was solved. Analysis of the anomalous scattering data obtained at two different wavelengths showed that zinc had displaced a significant proportion of iron from both iron centers of the diiron site, and that no iron had been displaced from the [Fe(SCys)4] site. UV–visible absorption spectra of the redissolved Zn,FeRbr crystals showed 30–40% retention of oxo-bridged diferric sites, and the redissolved crystals had 37% of the peroxidase specific activity of the starting all-iron Rbr, which, together with the crystallographic results, indicate a predominant mixture of Fe1,Fe2 and Zn1,Zn2 sites. The structure of the Zn(Fe)1,Fe(Zn)2 binuclear site in the Zn,FeRbr crystals was very similar to that of the Zn,Fe binuclear site reported for the “as-isolated” D. vulgaris Rbr, including tetrahedral four-coordination at the Zn(Fe)1 site. The diiron sites in the recombinant Zn,FeRbr crystals were likely at least partially reduced during synchrotron irradiation. Our results suggest that the mixed-metal binuclear site reported for the “as-isolated” D. vulgaris Rbr could be due to displacement of iron from a native diiron site by adventitious zinc during isolation and/or crystallization, and that reduced diiron and dizinc sites can adopt very similar structures in Rbr. [Copyright &y& Elsevier]

The thermostabilities of Fe2+ ligation in rubredoxins (Rds) from the hyperthermophile Pyrococcus furiosus (Pf) and the mesophiles Clostridium pasteurianum (Cp) and Desulfovibrio vulgaris (Dv) were compared. Residue 44 forms an NH...S(Cys) hydrogen bond to one of the cysteine ligands to the [Fe(SCys)4] site, and substitutions at this location affect the redox properties of the [Fe(SCys)4] site. Both Pf Rd and Dv Rd have an alanine residue at position 44, whereas Cp Fd has a valine residue. Wild-type proteins were examined along with V44A and A44V "exchange" mutants of Cp and Pf Rds, respectively, in order to assess the effects of the residue at position 44 on the stability of the [Fe(SCys)4] site. Stability of iron ligation was measured by temperature-ramp and fixed-temperature time course experiments, monitoring iron release in both the absence and presence of more thiophilic metals (Zn2+, Cd2+) and over a range of pH values. The thermostability of the polypeptide fold was concomitantly measured by fluorescence, circular dichroism, and 1H NMR spectroscopies. The A44V mutation strongly lowered the stability of the [FeII(SCys)4] site in Pf Rd, whereas the converse V44A mutation of Cp Rd significantly raised the stability of the [FeII(SCys)4] site, but not to the levels measured for wild-type Dv Rd. The region around residue 44 is thus a significant contributor to stability of iron coordination in reduced Rds. This region, however, made only a minor contribution to the thermostability of the protein folding, which was found to be higher for hyperthermophilic versus mesophilic Rds, and largely independent of the residue at position 44. These results, together with our previous studies, show that localized charge density, solvent accessibility, and iron site/backbone interactions control the thermostability of the [Fe(SCys)4] site. The iron site thermostability does make a minor contribution to the overall Rd thermostability. From a mechanistic standpoint, we also found that attack of displacing ions (H+, Cd2+) on the Cys42 sulfur ligand at the [Fe(SCys)4] site occurs through the V8 side and not the V44 side of the iron site. [ABSTRACT FROM AUTHOR]

Presents a study which indicates that an engineered two-iron superoxide reductase lacking the [Fe(SCys)[sub 4]] site retains its catalytic properties in vitro and in vivo. Physical, spectroscopic and redox characterization of C13S 2FeSOR; Complementation of E. coli by C13S 2Fe-SOR; Pulse radiolysis.

Examines the X-ray crystal structure of the recombinant rubrerythrin (Rbr) protein from the sulfate-reducing bacteria Desulfovibrio vulgaris. Structure of its azide adduct; Reduction of hydrogen peroxide by Rbr; Hydrogen bonding.

Reported are the X-ray crystal structures of recombinant Phascolopsis gouldii methemerythrin (1.8-Å resolution) and the structure of an O2-binding-pocket mutant, L98Y methemerythrin (2.1-Å resolution). The L98Y hemerythrin (Hr) has a greatly enhanced O2 affinity, a slower O2 dissociation rate, a larger solvent deuterium isotope effect on this rate, and a greater resistance to autoxidation relative to the wild-type protein. The crystal structures show that the hydrophobic binding pocket of Hr can accommodate substitution of a leucyl by a tyrosyl side chain with relatively minor structural rearrangements. UV/vis and resonance Raman spectra show that in solution L98Y methemerythrin contains a mixture of two diiron site structures differing by the absence or presence of an Fe(III)-coordinated phenolate. However, in the crystal, only one L98Y diiron site structure is seen, in which the Y98 hydroxyl is not a ligand, but instead forms a hydrogen bond to a terminal hydroxo/aqua ligand to the nearest iron. Based on this crystal structure, we propose that in the oxy form of L98Y hemerythrin the non-polar nature of the binding pocket favors localization of the Y98 hydroxyl near the O2 binding site, where it can donate a hydrogen bond to the hydroperoxo ligand. The stabilizing Y98OH-O2H– interaction would account for all of the altered O2 binding properties of L98Y Hr listed above. [ABSTRACT FROM AUTHOR]

Focuses on the immunoblotting of Desulfovibrio chemoreceptor genes expressed in a hemerythrin (Hr)-like domain. Presence of Hr protein in some marine invertebrates; Evidence of four-helix bundle structure in the C-terminal Hr-like domain; Discovery of Hr-like sequence motif at the C-terminus of the putative protein from a sulfate-reducing bacterium.

The single Fe(II) in reduced rubredoxin from Clostridium pasteurianum was found to be quantitatively displaced by either Cd2+ or Zn2+ when a modest molar excess of the substituting metal salt was anaerobically incubated with the reduced rubredoxin under mild conditions, namely, room temperature, pH 5.4–8.4, and no protein denaturants. Under the same conditions, cadmium-for-zinc substitution was also achieved upon aerobic incubation of the zinc-substituted rubredoxin with a modest molar excess of Cd2+. Displacements of Fe(II) from the reduced rubredoxin were not observed upon anaerobic incubation with Ni2+, Co2+, or VO2+ salts, and no reaction with any of the divalent metal ions was observed for the oxidized [Fe(III)] rubredoxin. Fe(II) could not be re-inserted into the Zn- or Cd-substituted rubredoxins without resorting to protein denaturation. 1H and 113Cd NMR experiments showed that the cadmium-substituted rubredoxin prepared by the non-denaturing substitution method retained the pseudotetrahedral M(SCys)4 coordination geometry and secondary structural elements characteristic of the native rubredoxin, and that "unzipping" of the β-sheet did not occur during metal substitution. Rates of Fe(II) displacement by M2+ (M=Cd or Zn) increased with increasing M2+/rubredoxin ratio, decreasing pH, and lower ionic strength. The substitution rates were faster for M=Cd than for M=Zn. Rates of Cd2+ substitution into a V8A-mutated rubredoxin were significantly faster than for the wild-type protein. The side-chain of V8 is on the protein surface and close to the metal-ligating Cys42Sγ at the M(SCys)4 site. Therefore, the rate-limiting step in the substitution process is suggested to involve direct attack of the [M(SCys)4]2– site by the incoming M2+, without global unfolding of the protein. Implications of these results for metal ion incorporation into rubredoxins in vivo are discussed. [ABSTRACT FROM AUTHOR]

Describes the synthesis and characterization of copper complexes of tris(imidazolyl)phosphine ligands containing 1,4-disubstituted imidazolyl groups and the reactions of the complexes with O2. Crystallization from acetontrile; Result of exposure of dry solutions of Cu1 complexes to O2 below -60 degrees centigrade.

Ex novo enzymic synthesis of the two 4Fe-4S clusters of Clostridium pasteurianum ferredoxin has been achieved by incubation of the apoprotein with catalytic amounts of the sulfurtransferase rhodanese in the presence of thiosulfate, DL-dihydrolipoate and ferric ammonium citrate. This enzymic reconstitution procedure was compared to a chemical one, in which the enzyme was replaced by sodium sulfide. A further comparison was made with the results previously obtained in the enzymic synthesis of the 2Fe-2S cluster of spinach ferredoxin, allowing the following conclusions to be drawn. (1) The nature of the duster to be inserted into the reconstituted iron-sulfur protein is determined by the apoprotein itself. (2) The refolding of the structure of the iron-sulfur proteins around the newly inserted cluster is the rate-limiting step in both chemical and enzymic reconstitution. (3) Rhodanese appears to play a role in the recovery of the native architecture of the reconstituted iron-sulfur protein(s). The extension to the 4Fe-4S centers of the rhodanese-based biosynthetic system allows this enzymic route to be proposed as a general way to the in vivo synthesis of iron-sulfur structures. [ABSTRACT FROM AUTHOR]