Pedro Tavares

Nitric Oxide Reductase (NOR) is an integral membrane protein performing the reduction of NO to N2O. 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 (TM)). 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.

Various S = 3/2 EPR signals elicited from wild-type and variant Azotobacter vinelandii nitrogenase MoFe proteins appear to reflect different conformations assumed by the FeMo-cofactor with different protonation states. To determine whether these presumed changes in protonation and conformation reflect catalytic capacity, the responses (particularly to changes in electron flux) of the alpha H195Q, alpha H195N, and alpha Q191 K variant MoFe proteins (where His at position 195 in the alpha subunit is replaced by Gln/Asn or Gln at position alpha-191 by Lys), which have strikingly different substrate-reduction properties, were studied by stopped-flow or rapid-freeze techniques. Rapid-freeze EPR at low electron flux (at 3-fold molar excess of wild-type Fe protein) elicited two transient FeMo-cofactor-based EPR signals within 1 s of initiating turnover under N-2 with the alpha H195Q and alpha H195N variants, but not with the alpha Q191K variant. No EPR signals attributable to P cluster oxidation were observed for any of the variants under these conditions. Furthermore, during turnover at low electron flux with the wild-type, alpha H195Q or alpha H195N MoFe protein, the longer-time 430-nm absorbance increase, which likely reflects P cluster oxidation, was also not observed (by stopped-flow spectrophotometry); it did, however, occur for all three MoFe proteins under higher electron flux. No 430-nm absorbance increase occurred with the alpha Q191K variant, not even at higher electron flux. This putative lack of involvement of the P cluster in electron transfer at low electron flux was confirmed by rapid-freeze Fe-57 Mossbauer spectroscopy, which clearly showed FeMo-factor reduction without P cluster oxidation. Because the wild-type, alpha H195Q and alpha H195N MoFe proteins can bind N-2, but alpha Q195K cannot, these results suggest that P cluster oxidation occurs only under high electron flux as required for N-2 reduction. (C) 2007 Elsevier Inc. All rights reserved.

The structure of desulfoferrodoxin (DFX), a protein containing two mononuclear non-heme iron centres, has been solved by the MAD method using phases determined at 2.8 Angstrom resolution. The iron atoms in the native protein were used as the anomalous scatterers. The model was built from an electron density map obtained after density modification and refined against data collected at 1.9 Angstrom. Desulfoferrodoxin is a homodimer which can be described in terms of two domains, each with two crystallographically equivalent non-heme mononuclear iron centres. Domain I is similar to desulforedoxin with distorted rubredoxin-type centres, and domain II has iron centres with square pyramidal coordination to four nitrogens from histidines as the equatorial ligands and one sulfur from a cysteine as the axial ligand. Domain I in DFX shows a remarkable structural fit with the DX homodimer. Furthermore, three beta-sheets extending from one monomer to another in DFX, two in domain I and one in domain II, strongly support the assumption of DFX as a functional dimer. A calcium ion, indispensable in the crystallisation process, was assumed at the dimer interface and appears to contribute to dimer stabilisation. The C-terminal domain in the monomer has a topology fold similar to that of fibronectin III.

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. Mössbauer 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.

Superoxide reductases are enzymes involved in bacterial resistance to reactive oxygen species, catalyzing the reduction of superoxide anions to hydrogen peroxide. So far three structural classes have been identified. Class I enzymes have two iron-center containing domains. Most studies have been focused on the catalytic iron site (center II), but the role of center I is yet poorly understood. The possible roles of this iron site were approached by an integrated study using both classical and fast kinetics measurements as well as direct electrochemistry. A new heterometallic form of the protein with a zinc-substituted center I, maintaining the iron active site center 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 2.8x107 (M-1s-1) and 1.3x106 (M-1s-1) for SORFe(IIII)-Fe(II) and for SORFe(IIII)-Fe(III) forms respectively, and 3.2x106 (M-1s-1) for the SORZn(II)-Fe(III) form. The results obtained seem to indicate that center I transfers electrons from the putative physiologic donor, rubredoxin, to the catalytic active iron site (intramolecular process). In addition, electrochemical results show that conformational changes are associated to the redox state of center 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.

{Crystals of the fully oxidized form of desulfoferrodoxin were obtained by vapor diffusion from a solution containing 20% PEG 4000, 0.1 M HEPES buffer, pH 7.5, and 0.2 M CaCl2. Trigonal and/or rectangular prisms could be obtained, depending on the temperature used for the crystal growth. Trigonal prisms belong to the rhombohedral space group R32, with a = 112.5 A and c = 63.2 A; rectangular prisms belong to the monoclinic space group C2, with a = 77.7 A

Ferrochelatase (EC 4.99.1.1) is the terminal enzyme of the haem biosynthetic pathway and catalyses iron chelation into the protoporphyrin IX ring. Glutamate-287 (E287) of murine mature ferrochelatase is a conserved residue in all known sequences of ferrochelatase, is present at the active site of the enzyme, as inferred from the Bacillus subtilis ferrochelatase three-dimensional structure, and is critical for enzyme activity. Substitution of E287 with either glutamine (Q) or alanine (A) yielded variants with lower enzymic activity than that of the wild-type ferrochelatase and with different absorption spectra from the wild-type enzyme. In contrast to the wild-type enzyme, the absorption spectra of the variants indicate that these enzymes, as purified, contain protoporphyrin IX. Identification and quantification of the porphyrin bound to the E287-directed variants indicate that approx. 80% of the total porphyrin corresponds to protoporphyrin IX. Significantly, rapid stopped-flow experiments of the E287A and E287Q Variants demonstrate that reaction with Zn2+ results in the formation of bound Zn-protoporphyrin IX, indicating that the endogenously bound protoporphyrin IX can be used as a substrate. Taken together, these findings suggest that the structural strain imposed by ferrochelatase on the porphyrin substrate as a critical step in the enzyme catalytic mechanism is also accomplished by the E287A and E287Q variants, but without the release of the product. Thus E287 in murine ferrochelatase appears to be critical For the catalytic process by controlling the release of the product.

Reactive oxygen species (ROS), when in excess, are among the most deleterious species an organism can deal with. The physiological effects of ROS include amino acid chain cleavage, DNA degradation and lipid oxidation, among others. They can be formed in the cytoplasm in a variety of ways, including autooxidation reactions (FMN- and FAD-containing enzymes) and Fenton reactions as a result of the cytoplasmatic pool of iron ions. The superoxide anion (021, despite its short half-life in solution, is particularly pernicious as it can form other reactive ROS (such as the strong oxidant peroxynitrite) or oxidize and/or reduce cellular components. For strict anaerobic or microaerophilic bacteria it is of particular importance to be able to dispose of ROS in a controlled manner, especially if these organisms are temporarily exposed to air. This review aims to describe the structural characteristics of superoxide reductases (SORs) and mechanistic aspects of biological superoxide anion reduction. SORs can be considered the main class of enzymes behind the oxygen detoxification pathway of anaerobic and microaerophilic bacteria. The geometry of the active site (three classes have been described), the possible electron donors in vivo and the current hypothesis for the catalytic mechanism will be discussed. Some phylogenetic considerations are presented, regarding the primary structure of SORs currently available in genome databases. ((c) Wiley-VCH Verlag GmbH {&} Co. KGaA, 69451 Weinheim, Germany, 2007).