The multicopper enzyme nitrous oxide reductase (N2OR) catalyzes the final step of denitrification, the two-electron reduction of N2O to N-2. This enzyme is a functional homodimer containing two different multicopper sites: CuA and CuZ. CuA is a binuclear copper site that transfers electrons to the tetranuclear copper sulfide CuZ, the catalytic site. In this study, Pseudomonas nautica cytochrome C-552 was identified as the physiological electron donor. The kinetic data show differences when physiological and artificial electron donors are compared [cytochrome vs methylviologen (MV)]. In the presence of cytochrome c(552), the reaction rate is dependent on the ET reaction and independent of the N2O concentration. With MV, electron donation is faster than substrate reduction. From the study of cytochrome c(552) concentration dependence, we estimate the following kinetic parameters: K-mc512 = 50.2 +/- 9.0 mu M and V-maxc551 1.8 +/- 10.6 units/mg. The N2O concentration dependence indicates a K-mN2O of 14.0 +/- 2.9 mu M using MV as the electron donor. The pH effect on the kinetic parameters is different when MV or cytochrome c(552) is used as the electron donor (pK(a) = 6.6 or 8.3, respectively). The kinetic study also revealed the hydrophobic nature of the interaction, and direct electron transfer studies showed that CuA is the center that receives electrons from the physiological electron donor. The formation of the electron transfer complex was observed by H-1 NMR protein-protein titrations and was modeled with a molecular docking program (BiGGER). The proposed docked complexes corroborated the ET studies giving a large number of solutions in which cytochrome c(552) is placed near a hydrophobic patch located around the CuA center.

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.

Superoxide reductases catalyze the monovalent reduction of superoxide anion to hydrogen peroxide. Spectroscopic evidence for the formation of a dinuclear cyano-bridged adduct after K3Fe-(CN)(6) oxidation of the superoxide reductases neelaredoxin from Treponema pallidum and desulfoferrodoxin from Desulfovibrio vulgaris was reported. Oxidation with K3Fe(CN)(6) reveals a band in the near-IR with lambda(max) at 1020 nm, coupled with an increase of the iron content by almost 2-fold. Fourier transform infrared spectroscopy provided additional evidence with CN-stretching vibrations at 2095, 2025-2030, and 2047 cm(-1), assigned to a ferrocyanide adduct of the enzyme. Interestingly, the low-temperature electronic paramagnetic resonance (EPR) spectra of oxidized TpNIr reveal at least three different species indicating structural heterogeneity in the coordination environment of the active site Fe ion. Given the likely 6-coordinate geometry of the active site Fe3+ ion in the ferrocyanide adduct, we propose that the rhombic EPR species can serve as a model of a hexacoordinate form of the active site.

The isolation and characterization of a new metalloprotein containing Cu and Fe atoms is reported. The as-isolated Cu-Fe protein shows an UV-visible spectrum with absorption bands at 320 nm, 409 nm and 615 nm. Molecular mass of the native protein along with denaturating electrophoresis and mass spectrometry data show that this protein is a multimer consisting of 14 +/- 1 subunits of 15254.3 +/- 7.6 Da. Mossbauer spectroscopy data of the as-isolated Cu-Fe protein is consistent with the presence of [2Fe-2S](2+) centers. Data interpretation of the dithionite reduced protein suggest that the metallic cluster could be constituted by two ferromagnetically coupled [2Fe-2S](+) spin delocalized pairs. The biochemical properties of the Cu-Fe protein are similar to the recently reported molybdenum resistance associated protein from Desulfovibrio, D. alaskensis. Further-more, a BLAST search from the DNA deduced amino acid sequence shows that the Cu-Fe protein has homology with proteins annotated as zinc resistance associated proteins from Desulfovibrio, D. alaskensis, D. vulgaris Hildenborough, D. piger ATCC 29098. These facts suggest a possible role of the Cu-Fe protein in metal tolerance. (C) 2009 Published by Elsevier Inc.