We report the 98% assignment of the apo-form of an orange protein, containing a novel Mo-Cu cluster isolated from Desulfovibrio gigas. This protein presents a region where backbone amide protons exchange fast with bulk solvent becoming undetectable. These residues were assigned using C-13-detection experiments.

Nitrous oxide (N(2)O) is a greenhouse gas, the third most significant contributor to global warming. As a key process for N(2)O elimination from the biosphere, N(2)O reductases catalyze the two-electron reduction of N(2)O to N(2). These 2 x 65 kDa copper enzymes are thought to contain a CuA electron entry site, similar to that of cytochrome c oxidase, and a CuZ catalytic center. The copper anomalous signal was used to solve the crystal structure of N(2)O reductase from Pseudomonas nautica by multiwavelength anomalous dispersion, to a resolution of 2.4 Angstrom. The structure reveals that the CuZ center belongs to a new type of metal cluster, in which four copper ions are liganded by seven histidine residues. N(2)O binds to this center via a single copper ion. The remaining copper ions might act as an electron reservoir, assuring a fast electron transfer and avoiding the formation of dead-end products.

Several studies have sought to understand the health effects of occupational exposure to cosmic radiation. However, only few biologic markers or associations with disease outcomes have so far been identified. In the present study, 22 long- and 26 medium-haul male Portuguese airline pilots and 36 factory workers who did not fly regularly were investigated. The two groups were comparable in age and diet, were non-smokers, never treated with ionizing radiation and other factors. Cosmic radiation exposure in pilots was quantified based on direct monitoring of 51 flights within Europe, and from Europe to North and South America, and to Africa. Indirect dose estimates in pilots were performed based on the SIEVERT (Système informatisé d'évaluation par vol de l'exposition au rayonnement cosmique dans les transports aériens) software for 6,039 medium- and 1,366 long-haul flights. Medium-haul pilots had a higher cosmic radiation dose rate than long-haul pilots, that is, 3.3 ± 0.2 μSv/h and 2.7 ± 0.3 μSv/h, respectively. Biological tests for oxidative stress on blood and urine, as appropriate, at two time periods separated by 1 year, included measurements of antioxidant capacity, total protein, ferritin, hemoglobin, creatinine and 8-hydroxy-2-deoxyguanosine (8OHdG). Principal components analysis was used to discriminate between the exposed and unexposed groups based on all the biological tests. According to this analysis, creatinine and 8OHdG levels were different for the pilots and the unexposed group, but no distinctions could be made among the medium- and the long-haul pilots. While hemoglobin levels seem to be comparable between the studied groups, they were directly correlated with ferritin values, which were lower for the airline pilots.

Superoxide reductases are a class of non-haem iron enzymes which catalyse the monovalent reduction of the superoxide anion O-2(-) into hydrogen peroxide and water. Treponema pallidum (Tp), the syphilis spirochete, expresses the gene for a superoxide reductase called neelaredoxin, having the iron protein rubredoxin as the putative electron donor necessary to complete the catalytic cycle. In this work, we present the first cloning, overexpression in Escherichia coli and purification of the Tp rubredoxin. Spectroscopic characterization of this 6 Da protein allowed us to calculate the molar absorption coefficient of the 490 nm feature of ferric iron, epsilon=6.9+/-0.4 mM(-1) cm(-1). Moreover, the midpoint potential of Tp rubredoxin, determined using a glassy carbon electrode, was -76+/-5 mV. Reduced rubredoxin can be efficiently reoxidized upon addition of Na2IrCl6-oxidized neelaredoxin, in agreement with a direct electron transfer between the two proteins, with a stoichiometry of the electron transfer reaction of one molecule of oxidized rubredoxin per one molecule of neelaredoxin. In addition, in presence of a steady-state concentration of superoxide anion, the physiological substrate of neelaredoxin, reoxidation of rubredoxin was also observed in presence of catalytic amounts of superoxide reductase, and the rate of rubredoxin reoxidation was shown to be proportional to the concentration of neelaredoxin, in agreement with a bimolecular reaction, with a calculated k(app)=180 min(-1). Interestingly, similar experiments performed with a rubredoxin from the sulfate-reducing bacteria Desulfovibrio vulgaris resulted in a much lower value of k(app)=4.5 min(-1). Altogether, these results demonstrated the existence for a superoxide-mediated electron transfer between rubredoxin and neelaredoxin and confirmed the physiological character of this electron transfer reaction.