Alice S. Pereira

An orange-coloured protein (ORP) isolated from Desulfovibrio gigas, a sulphate reducer, has been previously shown by extended X-ray absorption fine structure (EXAFS) to contain a novel mixed-metal sulphide cluster of the type [S2MoS2CuS2MoS2] [J. Am. Chem. Soc. 122 (2000) 8321]. We report here the purification and the biochemical/spectroscopic characterisation of this novel protein. ORP is a soluble monomeric protein (11.8 kDa). The cluster is non-covalently bound to the polypeptide chain. The presence of a MoS42- moiety in the structure of the cofactor contributes with a quite characteristic UV-Vis spectra, exhibiting an orange colour, with intense absorption peaks at 480 and 338 nm. Pure ORP reveals an Abs(480)/Abs(338) ratio of 0.535. The gene sequence coding for ORP as well as the amino acid sequence was determined. The putative biological function of ORP is discussed. (C) 2003 Elsevier Inc. All rights reserved.

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 (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.

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.

Respiratory nitric oxide reductase (NOR) was purified from membrane extract of Pseudomonas (Ps.) nautica cells to homogeneity as judged by polyacrylamide gel electrophoresis. The purified protein is a heterodimer with subunits of molecular masses of 54 and 18 kDa. The gene encoding both subunits was cloned and sequenced. The amino acid sequence shows strong homology with enzymes of the cNOR class. Iron/heme determinations show that one heme c is present in the small subunit (NORC) and that approximately two heme b and one non-heme iron are associated with the large subunit (NORB), in agreement with the available data for enzymes of the cNOR class. Mossbauer characterization of the as-purified, ascorbate-reduced, and dithionite-reduced enzyme confirms the presence of three heme groups (the catalytic heme b(3) and the electron transfer heme b and heme c) and one redox-active non-heme Fe (Fe-B). Consistent with results obtained for other cNORs, heme c and heme b in Ps. nautica cNOR were found to be low-spin while FeB was found to be high-spin. Unexpectedly, as opposed to the presumed high-spin state for heme b(3), the Mossbauer data demonstrate unambiguously that heme b(3) is, in fact, low-spin in both ferric and ferrous states, suggesting that heme b(3) is six-coordinated regardless of its oxidation state. EPR spectroscopic measurements of the as-purified enzyme show resonances at the g similar to 6 and g similar to 2-3 regions very similar to those reported previously for other cNORs. The signals at g = 3.60, 2.99, 2.26, and 1.43 are attributed to the two charge-transfer low-spin ferric heme c and heme b. Previously, resonances at the g similar to 6 region were assigned to a small quantity of uncoupled high-spin Fe-III heme b(3). This assignment is now questionable because heme b(3) is low-spin. On the basis of our spectroscopic data, we argue that the g = 6.34 signal is likely arising from a spin spin coupled binuclear center comprising the low-spin Fe-III heme b(3) and the high-spin Fe-B(III). Activity assays performed under various reducing conditions indicate that heme b(3) has to be reduced for the enzyme to be active. But, from an energetic point of view, the formation of a ferrous heme-NO as an initial reaction intermediate for NO reduction is disfavored because heme [FeNO](7) is a stable product. We suspect that the presence of a sixth ligand in the Fe-II-heme b(3) may weaken its affinity for NO and thus promotes, in the first catalytic step, binding of NO at the Fe-B(II) site. The function of heme b(3) would then be to orient the Fe-B-bound NO molecules for the formation of the N-N bond and to provide reducing equivalents for NO reduction.

The characterization of a novel Mo-Fe protein (MorP) associated with a system that responds to Mo in Desulfovibrio alaskensis is reported. Biochemical characterization shows that MorP is a periplasmic homomultimer of high molecular weight (260 +/- 13 kDa) consisting of 16-18 monomers of 15321.1 +/- 0.5 Da. The UV/visible absorption spectrum of the as-isolated protein shows absorption peaks around 280, 320, and 570 nm with extinction coefficients of 18700, 12800, and 5000 M(-1) cm(-1), respectively. Metal content, EXAFS data and DFT calculations support the presence of a Mo-2S-[2Fe-2S]-2S-Mo cluster never reported before. Analysis of the available genomes from Desulfovibrio species shows that the MorP encoding gene is located downstream of a sensor and a regulator gene. This type of gene arrangement, called two component system, is used by the cell to regulate diverse physiological processes in response to changes in environmemtal conditions. Increase of both gene expression and protein production was observed when cells were cultured in the presence of 45 mu M molybdenum. Involvement of this system in Mo tolerance of sulfate reducing bacteria is proposed.

Adenylate kinase (AK) mediates the reversible transfer of phosphate groups between the adenylate nucleotides and contributes to the maintenance of their constant cellular level, necessary for energy metabolism and nucleic acid synthesis. The AK were purified from crude extracts of two sulfate-reducing bacteria (SRB), Desulfovibrio (D.) gigas NCIB 9332 and Desulfovibrio desulfuricans ATCC 27774, and biochemically and spectroscopically characterised in the native and fully cobalt- or zinc-substituted forms. These are the first reported adenylate kinases that bind either zinc or cobalt and are related to the subgroup of metal-containing AK found, in most cases, in Gram-positive bacteria. The electronic absorption spectrum is consistent with tetrahedral coordinated cobalt, predominantly via sulfur ligands, and is supported by EPR. The involvement of three cysteines in cobalt or zinc coordination was confirmed by chemical methods. Extended X-ray absorption fine structure (EXAFS) indicate that cobalt or zinc are bound by three cysteine residues and one histidine in the metal-binding site of the "LID" domain. The sequence (129)Cys-X(5)-His-X(15)-Cys-X(2)-Cys of the AK from D. gigas is involved in metal coordination and represents a new type of binding motif that differs from other known zinc-binding sites of AK. Cobalt and zinc play a structural role in stabilizing the LID domain. (C) 2008 Elsevier Inc. All rights reserved.

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.

The gene for pseudoazurin was isolated from Paracoccus pantotrophus LMD 52.44 and expressed in a heterologous system with a yield of 54.3 mg of pure protein per liter of culture. The gene and protein were shown to be identical to those from P. pantotrophus LMD 82.5. The extinction coefficient of the protein was re-evaluated and was found to be 3.00 mM(-1) cm(-1) at 590 nm. It was confirmed that the oxidized protein is in a weak monomer/dimer equilibrium that is ionic- strength-dependent. The pseudoazurin was shown to be a highly active electron donor to cytochrome c peroxidase, and activity showed an ionic strength dependence consistent with an electrostatic interaction. The pseudoazurin has a very large dipole moment, the vector of which is positioned at the putative electron-transfer site, His81, and is conserved in this position across a wide range of blue copper proteins. Binding of the peroxidase to pseudoazurin causes perturbation of a set of NMR resonances associated with residues on the His81 face, including a ring of lysine residues. These lysines are associated with acidic residues just back from the rim, the resonances of which are also affected by binding to the peroxidase. We propose that these acidic residues moderate the electrostatic influence of the lysines and so ensure that specific charge interactions do not form across the interface with the peroxidase.

An air-stable formate dehydrogenase (FDH), an enzyme that catalyzes the oxidation of formate to carbon dioxide, was purified from the sulfate reducing organism Desulfovibrio gigas (D. gigas) NCIB 9332. D. gigas FDH is a heterodimeric protein [alpha (92 kDa) and beta (29 kDa) subunits] and contains 7 +/- 1 Fe/protein and 0.9 +/- 0.1 W/protein, Selenium was not detected. The UV/visible absorption spectrum of D, gigas FDH is typical of an iron-sulfur protein. Analysis of pterin nucleotides yielded a content of 1.3 +/- 0.1 guanine monophosphate/mol of enzyme, which suggests a tungsten coordination with two molybdopterin guanine dinucleotide cofactors. Both Mossbauer spectroscopy performed on D. gigas FDH grown in a medium enriched with Fe-57 and EPR studies performed in the native and fully reduced state of the protein confirmed the presence of two [4Fe-4S] clusters. Variable-temperature EPR studies showed the presence of two signals compatible with an atom in a d(1) configuration albeit with an unusual relaxation behavior as compared to the one generally observed for W(V) ions.

The aerobic purification of Pseudomonas nautica 617 nitrous oxide reductase yielded two forms of the enzyme exhibiting different chromatographic behaviors. The protein contains six copper atoms per monomer, arranged in two centers named CUA and Cut. Cut could be neither oxidized nor further reduced under our experimental conditions, and exhibits a 4-line EPR spectrum (g(x)= 2.015, A(x) = 1.5 mT, g(y) = 2.071, A(y) = 2 mT, g(z) = 2.138, A(z) = 7 mT) and a strong absorption at similar to 640 nm. Cu-A can be stabilized in a reduced EPR-silent state and in an oxidized state with a typical 7-line EPR spectrum (g(x) g(y) = 2.021, A(x) = A(y) = 0 T, g(z) =0.178, A(z) = 4 mT) and absorption bands at 480, 540, and similar to 800 nm. The difference between the two purified forms of nitrous oxide reductase is interpreted as a difference in the oxidation state of the CuA center. In form A, CUA is predominantly oxidized (S = 1/2, Cu1.5+-Cu1.5+), while in form B it is mostly in the one-electron reduced state (S = 0, Cu1+-Cu1+). In both forms, Cu-Z remains reduced (S = 1/2). Complete crystallographic data at 2.4 Angstrom indicate that Cu-A is a binuclear site (similar to the site found in cytochrome c oxidase) and Cu-Z is a novel tetracopper cluster [Brown, K., et ai. (2000) Nat. Struct. Biol. (in press)]. The complete amino acid sequence of the enzyme was determined and comparisons made with sequences of other nitrous oxide reductases, emphasizing the coordination of the centers. A 10.3 kDa peptide copurified with both forms of nitrous oxide reductase shows strong homology with proteins of the heat-shock GroES chaperonin family.

Conversion of Fe ions in solution to the solid phase in ferritin concentrates iron required for cell function. The rate of the Fe phase transition in ferritin is tissue specific and reflects the differential expression of two classes of ferritin subunits (H and L). Early stages of mineralization were probed by rapid freeze-quench Mossbauer, at strong fields (up to 8 T), and EPR spectroscopy in an H-type subunit, recombinant frog ferritin; small numbers of Fe (36 moles/mol of protein) were used to increase Fe3+ in mineral precursor forms, At 25 ms, four Fe3+-oxy species (three Fe dimers and one Fe trimer) were identified, These Fe3+-oxy species were found to form at similar rates and decay subsequently to a distinctive superparamagentic species designated the ''young core.'' The rate of oxidation of Fe2+ (1026 s(-1)) corresponded well to the formation constant for the Fe3+- tyrosinate complex (920 s(-1)) observed previously [Waldo, G. S., & Theil, E. C. (1993) Biochemistry 32, 13261] and, coupled with EPR data, indicates that several or possibly all of the Fe3+-oxy species involve tyrosine. The results, combined with previous Mossbauer studies of Y30F human H-type ferritin which showed decreases in several Fe3+ intermediates and stabilization of Fe2+ [Bauminger, E. R., et al. (1993) Biochem, J. 296, 709], emphasize the involvement of tyrosyl residues in the mineralization of H-type ferritins. The subsequent decay of these multiple Fe3+-oxy species to the superparamagnetic mineral suggests that Fe3+ species in different environments may be translocated as intact units from the protein shell into the ferritin cavity where the conversion to a solid mineral occurs.

Ferritins are ubiquitous and can be found in practically all organisms that utilize Fe. They are composed of 24 subunits forming a hollow sphere with an inner cavity of similar to 80 angstrom in diameter. The main function of ferritin is to oxidize the cytotoxic Fe2+ ions and store the oxidized Fe in the inner cavity. It has been established that the initial step of rapid oxidation of Fe2+ (ferroxidation) by H-type ferritins, found in vertebrates, occurs at a diiron binding center, termed the ferroxidase center. In bacterial ferritins, however, X-ray crystallographic evidence and amino acid sequence analysis revealed a trinuclear Fe binding center comprising a binuclear Fe binding center (sites A and B), homologous to the ferroxidase center of H-type ferritin, and an adjacent mononuclear Fe binding site (site C). In an effort to obtain further evidence supporting the presence of a trinuclear Fe binding center in bacterial ferritins and to gain information on the states of the iron bound to the trinuclear center, bacterial ferritin from Desulfovibrio vulgaris (DvFtn) and its E130A variant was loaded with substoichiometric amounts of Fe2+, and the products were characterized by Mossbauer and EPR spectroscopy. Four distinct Fe species were identified: a paramagnetic diferrous species, a diamagnetic diferrous species, a mixed valence Fe2+Fe3+ species, and a mononuclear Fe2+ species. The latter three species were detected in the wild-type DvFtn, while the paramagnetic diferrous species was detected in the E130A variant. These observations can be rationally explained by the presence of a trinuclear Fe binding center, and the four Fe species can be properly assigned to the three Fe binding sites. Further, our spectroscopic data suggest that (1) the fully occupied trinuclear center supports an all ferrous state, (2) sites B and C are bridged by a mu-OH group forming a diiron subcenter within the trinuclear center, and (3) this subcenter can afford both a mixed valence Fe2+Fe3+ state and a diferrous state. Mechanistic insights provided by these new findings are discussed and a minimal mechanistic scheme involving O-O bond cleavage is proposed.

Cytochrome c peroxidase (CCP) catalyses the reduction of H2O2 to H2O, an important step in the cellular detoxification process. The crystal structure of the di-heme CCP from Pseudomonas nautica 617 was obtained in two different conformations in a redox state with the electron transfer heme reduced. Form IN, obtained at pH 4.0, does not contain Ca2+ and was refined at 2.2 Angstrom resolution. This inactive form presents a closed conformation where the peroxidatic heme adopts a six-ligand coordination, hindering the peroxidatic reaction from taking place. Form OUT is Ca2+ dependent and was crystallized at pH 5.3 and refined at 2.4 Angstrom resolution. This active form shows an open conformation, with release of the distal histidine (His71) ligand, providing peroxide access to the active site. This is the first time that the active and inactive states are reported for a di-heme peroxidase.