SORs (superoxide reductases) are enzymes involved in bacterial resistance to reactive oxygen species, catalysing the reduction of superoxide anions to hydrogen peroxide. So far three structural classes have been identified. Class I enzymes have two iron-centre-containing domains. Most studies have focused on the catalytic iron site (centre II), yet the role of centre I is poorly understood. The possible roles of this iron site were approached by an integrated study using both classical and fast kinetic measurements, as well as direct electrochemistry. A new heterometallic form of the protein with a zinc-substituted centre I, maintaining the iron active-site centre II, was obtained, resulting in a stable derivative useful for comparison with the native all-iron from. Second-order rate constants for the electron transfer between reduced rubredoxin and the different SOR forms were determined to be 2.8 x 10 M(1) . s(1) and 1.3 x 10 M(1) . s(1) for SORFe(IIII)-Fe(II) and for SORFe(IIII)-Fe(III) forms respectively, and 3.2 x 10 M(1) . s(1) for the SORZn(II)-Fe(III) form. The results obtained seem to indicate that centre I transfers electrons from the putative physiological donor rubredoxin to the catalytic active iron site (intramolecular process). In addition, electrochemical results show that conformational changes are associated with the redox state of centre I, which may enable a faster catalytic response towards superoxide anion. The apparent rate constants calculated for the SOR-mediated electron transfer also support this observation.

Analysis of sequenced bacterial genomes revealed that the genomes encode more than 30% hypothetical and conserved hypothetical proteins of unknown function. Among proteins of unknown function that are conserved in anaerobes, some might be determinants of the anaerobic way of life. This study focuses on two divergent clusters specifically found in anaerobic microorganisms and mainly composed of genes encoding conserved hypothetical proteins. We show that the two gene clusters DVU2103-DVU2104-DVU2105 (orp2) and DVU2107-DVU2108-DVU2109 (orp1) form two divergent operons transcribed by the sigma(54)-RNA polymerase. We further demonstrate that the sigma(54)-dependent transcriptional regulator DVU2106, located between orp1 and orp2, collaborates with sigma(54)-RNA polymerase to orchestrate the simultaneous expression of the divergent orp operons. DVU2106, whose structural gene is transcribed by the sigma(70)-RNA polymerase, negatively retrocontrols its own expression. By using an endogenous pulldown strategy, we identify a physiological complex composed of DVU2103, DVU2104, DVU2105, DVU2108, and DVU2109. Interestingly, inactivation of DVU2106, which is required for orp operon transcription, induces morphological defects that are likely linked to the absence of the ORP complex. A putative role of the ORP proteins in positioning the septum during cell division is discussed.

The unfolding dynamics of the rubredoxin mutant A51C (RdA51C) from Desulfovibrio vulgaris (DvRd) was studied on the temperature range from 25 degrees C to 90 degrees C and by incubation at 90 degrees C. By Forster Resonance Energy Transfer (FRET) the donor (D; Trp37) to acceptor (A; 1,5-IAEDANS) distance distribution was probed at several temperatures between 25 degrees C and 90 degrees C, and incubation times at 90 degrees C. From 25 degrees C to 50 degrees C the half-width distributions values (hw) are small and the presence of a discrete D-A distance was considered. At temperatures higher than 60 degrees C broader hw values were observed reflecting the existence of a distance distribution. The protein denaturation was only achieved by heating the solution for 2 h at 90 degrees C, as probed by the increase of the D-A mean distance. From Trp fluorescence it was shown that its vicinity was maintained until similar to 70 degrees C, being the protein hydrodynamic radius invariant until 50 degrees C. However, at similar to 70 degrees C a change in the partial unfolding kinetics indicates the disruption of specific H-bonds occurring in the hydrophobic core. The red shift of 13 nm, observed on the Trp37 emission, confirms the exposition of Trp to solvent after protein incubation at 90 degrees C for 2.5 h. (C) 2010 Elsevier B.V. All rights reserved.

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. Furthermore, 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.

Nitric Oxide Reductase (NOR) is an integral membrane protein performing the reduction of NO to N(2)O. NOR is composed of two subunits: the large one (NorB) is a bundle of 12 transmembrane helices (TMH). It contains a b type heme and a binuclear iron site, which is believed to be the catalytic site, comprising a heme b and a non-hemic iron. The small subunit (NorC) harbors a cytochrome c and is attached to the membrane through a unique TMH. With the aim to perform structural and functional studies of NOR, we have immunized dromedaries with NOR and produced several antibody fragments of the heavy chain (VHHs, also known as nanobodies). These fragments have been used to develop a faster NOR purification procedure, to proceed to crystallization assays and to analyze the electron transfer of electron donors. BIAcore experiments have revealed that up to three VHHs can bind concomitantly to NOR with affinities in the nanomolar range. This is the first example of the use of VHHs with an integral membrane protein. Our results indicate that VHHs are able to recognize with high affinity distinct epitopes on this class of proteins, and can be used as versatile and valuable tool for purification, functional study and crystallization of integral membrane proteins.

Aldehyde oxidoreductase from Desulfovibrio gigas (DgAOR) is a member of the xanthine oxidase (XO) family of mononuclear Mo-enzymes that catalyzes the oxidation of aldehydes to carboxylic acids. The molybdenum site in the enzymes of the XO family shows a distorted square pyramidal geometry in which two ligands, a hydroxyl/water molecule (the catalytic labile site) and a sulfido ligand, have been shown to be essential for catalysis. We report here steady-state kinetic studies of DgAOR with the inhibitors cyanide, ethylene glycol, glycerol, and arsenite, together with crystallographic and EPR studies of the enzyme after reaction with the two alcohols. In contrast to what has been observed in other members of the XO family, cyanide, ethylene glycol, and glycerol are reversible inhibitors of DgAOR. Kinetic data with both cyanide and samples prepared from single crystals confirm that DgAOR does not need a sulfido ligand for catalysis and confirm the absence of this ligand in the coordination sphere of the molybdenum atom in the active enzyme. Addition of ethylene glycol and glycerol to dithionite-reduced DgAOR yields rhombic Mo(V) EPR signals, suggesting that the nearly square pyramidal coordination of the active enzyme is distorted upon alcohol inhibition. This is in agreement with the X-ray structure of the ethylene glycol and glycerol-inhibited enzyme, where the catalytically labile OH/OH(2) ligand is lost and both alcohols coordinate the Mo site in a eta(2) fashion. The two adducts present a direct interaction between the molybdenum and one of the carbon atoms of the alcohol moiety, which constitutes the first structural evidence for such a bond in a biological system.

Membrane-bound nitrate reductase from Marinobacter hydrocarbonoclasticus 617 can be solubilized in either of two ways that will ultimately determine the presence or absence of the small (Iota) subunit. The enzyme complex (NarGHI) is composed of three subunits with molecular masses of 130, 65, and 20 kDa. This enzyme contains approximately 14 Fe, 0.8 Mo, and 1.3 molybdopterin guanine dinucleotides per enzyme molecule. Curiously, one heme b and 0.4 heme c per enzyme molecule have been detected. These hemes were potentiometrically characterized by optical spectroscopy at pH 7.6 and two noninteracting species were identified with respective midpoint potentials at Em=+197 mV (heme c) and -4.5 mV (heme b). Variable-temperature (4-120 K) X-band electron paramagnetic resonance (EPR) studies performed on both as-isolated and dithionite-reduced nitrate reductase showed, respectively, an EPR signal characteristic of a [3Fe-4S]+ cluster and overlapping signals associated with at least three types of [4Fe-4S]+ centers. EPR of the as-isolated enzyme shows two distinct pH-dependent Mo(V) signals with hyperfine coupling to a solvent-exchangeable proton. These signals, called "low-pH" and "high-pH," changed to a pH-independent Mo(V) signal upon nitrate or nitrite addition. Nitrate addition to dithionite-reduced samples at pH 6 and 7.6 yields some of the EPR signals described above and a new rhombic signal that has no hyperfine structure. The relationship between the distinct EPR-active Mo(V) species and their plausible structures is discussed on the basis of the structural information available to date for closely related membrane-bound nitrate reductases.

Three ultrasonic energy sources were studied to speed up the sample treatment for in-solution protein identification by peptide mass fingerprint using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Protein reduction, alkylation, and enzymatic digestion steps were done in 15 min. Nine proteins, including zinc resistance-associated protein precursor from Desulfovibrio desulfuricans strain G20 and split-soret cytochrome c from D. desulfuricans ATCC27774 were successfully identified with the new protocol.

Fast (120 s) and high-throughput (more than six samples at once) in-gel trypsin digestion of proteins using sonoreactor technology has been achieved. Successful protein identification was done by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, MALDI-TOF-MS. Specific identification of the adenylylsulphate reductase alfa subunit from a complex protein mixture from Desulfovibrio desulfuricans ATCC 27774 was done as a proof of the methodology. The new sample treatment is of easy implementation, saves time and money, and can be adapted to online procedures and robotic platforms.