An air-stable formate dehydrogenase, an enzyme that catalyzes the oxidation of formate to CO2, was purified from a sulfate-reducing organism, Desulfovibrio desulfuricans ATCC 27774. The enzyme has a molecular mass of approximately 150 kDa (three different subunits: 88, 29 and 16 kDa) and contains three types of redox-active centers: four c-type hemes, nonheme iron arranged as two [4Fe-4S](2+/1+) centers and a molybdenum-pterin site. Selenium was also chemically detected. The enzyme specific activity is 78 units per mg of protein. Mo(V) EPR signals were observed in the native, reduced and formate-reacted states. EPR signals related to the presence of multiple low-spin hemes were also observed in the oxidized state. Upon reduction, an examination of the EPR data under appropriate conditions distinguishes two types of iron-sulfur centers, an [Fe-S] center I (g(max)=2.050, g(med)=1.947, g(min)=1.896) and an [Fe-S] center II (g(max)=2.071, g(med)=1.926, g(min)=1.865). Mossbauer spectroscopy confirmed the presence of four hemes in the low-spin state. The presence of two [4Fe-4S](2+/1+) centers was confirmed, one of these displaying very small hyperfine coupling constants in the +1 oxidation state. The midpoint redox potentials of the enzyme metal centers were also estimated.

Metallothionein (MT) in the liver of gilthead seabreams (Sparus aurata L., 1758) exposed to Sado estuary (Portugal) sediments was quantified to assess the MT induction potential as a biomarker of sediment-based contamination by copper (Cu), cadmium (U), lead (Pb) and arsenic (As). Sediments were collected from two control sites and four sites with different levels of contamination. Sediment Cu, Cd, Pb, As, total organic matter (TOM) and fine fraction (FF) levels were determined. Generalized linear models (GLM) allowed integration of sediment parameters with liver Cu, Cd, Pb, As and MT concentrations. Although sediment metal levels were lower than expected, we relate NIT with liver Cd and also with interactions between liver and sediment Cu and between liver Cu and TOM. We suggest integrating biomarkers and environmental parameters using statistical models such as GLM as a more sensitive and reliable technique for sediment risk assessment than traditional isolated biomarker approaches. (C) 2007 Elsevier Inc. All rights reserved.

Dissimilatory nitrite reduction, carried out by hexaheme proteins, gives ammonia as the final product. Representatives of this enzyme group from 3 bacterial species can also reduce NO to either ammonia or N2O. The redox regulation of the nitrite/nitric oxide activities is discussed in the context of the denitrifying pathway.

The dissimilatory nitrite reductase from Desulfovibrio desulfuricans ATCC 27774 catalyzes the reduction of nitrite to ammonia. Previous spectroscopic investigation revealed that it is a hexaheme cytochrome containing one high spin ferric heme and five low spin ferric hemes in the oxidized enzyme. The current study uses the high resolution of Mossbauer spectroscopy to obtain redox properties of the six heme groups. Correlating the Mossbauer findings with the EPR data reveals the pairwise spin-spin coupling among four of the heme groups. The other two hemes are found to be magnetically isolated. Reduction with dithionite and reaction with CO further indicate that only the high spin heme is capable of binding small exogenous ligands. These results confirm our previous finding that Desulfovibrio desulfuricans nitrite reductase contains six heme groups and that the high spin ferric heme is the substrate and inhibitor binding site.

In humans, the lymphomyeloid system has a fundamental role on iron metabolism promoting its recycling due to a continuous removal of effete red blood cells. Additionally, one of the most intriguing aspects of metalloporphyrins in biology is their effect on the immune system. However, the process of erythrocyte catabolism is still poorly understood and needs further research. In the present study, we attempt to investigate the nature and the possible physiologic role of Fe compounds released after erythrophagocytosis during the removal of red blood cells. Monocyte erythrophagocytosis in vitro experiments were done to characterize chemically the Fe compounds present inside the cells and in the culture supernatants. We tested the probable immunomodulatory functions of erythrophagocytosis products over lymphocyte cultures activated in vitro with T mitogens (alpha-CD3). Data obtained from atomic absorption spectroscopy confirmed the presence of Fe in the culture supernatants of monocyte cultures after erythrophagocytosis. Also, high-spin haem complexes derived from erythrocyte catabolism were detected by electron paramagnetic electronic resonance. Finally, in vitro activated lymphocyte proliferation experiments indicate the co-mitogenic properties of monocyte culture supernatants after red blood cells phagocytosis. Thus, the results of the present work provide evidence that culture monocyte supernatants after in vitro erythrophagocytosis contain Fe (III) high-spin haem complexes and show lymphocyte proliferation co-stimulatory properties.

The molybdenum EXAFS of the Mo(2Fe-2S) protein from Desulfovibrio gigas has been examined using fluorescence detection and synchrotron radiation. In the oxidized form the molybdenum environment is found to contain two terminal oxo groups and two long (2.47 A) Mo-S bonds. Evidence was also found for an oxygen or nitrogen donor ligand at 1.90 A. Addition of dithionite to the oxidized enzyme results in loss of a terminal oxo group, perhaps due to protonation. In addition, a 0.1 A contraction in the Mo-S bond lengths is observed. The behavior of both oxidized and dithionite-treated forms is similar to that observed previously with "desulfo" xanthine oxidase.

The gene encoding cytochrome c nitrite reductase (NrfA) from Desulfovibrio desulfuricans ATCC 27774 was sequenced and the crystal structure of the enzyme was determined to 2.3-A resolution. In comparison with homologous structures, it presents structural differences mainly located at the regions surrounding the putative substrate inlet and product outlet, and includes a well defined second calcium site with octahedral geometry, coordinated to propionates of hemes 3 and 4, and caged by a loop non-existent in the previous structures. The highly negative electrostatic potential in the environment around hemes 3 and 4 suggests that the main role of this calcium ion may not be electrostatic but structural, namely in the stabilization of the conformation of the additional loop that cages it and influences the solvent accessibility of heme 4. The NrfA active site is similar to that of peroxidases with a nearby calcium site at the heme distal side nearly in the same location as occurs in the class II and class III peroxidases. This fact suggests that the calcium ion at the distal side of the active site in the NrfA enzymes may have a similar physiological role to that reported for the peroxidases.

The dsr gene from Desulfovibrio gigas encoding the nonheme iron protein desulforedoxin was cloned using the polymerase chain reaction, expressed in Escherichia coli, and purified to homogeneity. The physical and spectroscopic properties of the recombinant protein resemble those observed for the native protein isolated from D. gigas. These include an alpha 2 tertiary structure, the presence of bound iron, and absorbance maxima at 370 and 506 nm in the UV/visible spectrum due to ligand-to-iron charge transfer bands. Low temperature electron paramagnetic resonance studies confirm the presence of a high-spin ferric ion with g values of 7.7, 5.7, 4.1, and 1.8. Interestingly, E. coli produced two forms of desulforedoxin containing iron. One form was identified as a dimer with the metal-binding sites of both subunits occupied by iron while the second form contained equivalent amounts of iron and zinc and represents a dimer with one subunit occupied by iron and the second with zinc.