http://hdl.handle.net/10362/1532 Sat, 25 May 2013 18:06:55 GMT 2013-05-25T18:06:55Z http://hdl.handle.net/10362/8724 Title: X-ray crystal structure and EPR spectra of "arsenite-inhibited" Desulfovibriogigas aldehyde dehydrogenase: a member of the xanthine oxidase family Authors: Boer, D. Roeland; Thapper, Anders; Brondino, Carlos D.; Romão, Maria J.; Moura, José J. G. Abstract: X-ray crystallography has been used to determine the structure of arsenite-inhibited aldehyde dehydrogenase from Desulfovibrio gigas, a member of the xanthine oxidase family of mononuclear molybdenum enzymes. The structure shows an AsO3 moiety bound to the molybdenum atom of the active site through one of the oxygen atoms. A reduced sample of arsenite-inhibited aldehyde dehydrogenase has a Mo(V) signal that shows anisotropic hyperfine and quadrupole coupling to one arsenic atom. This signal has a strong resemblance with a previously reported signal for arsenite-inhibited xanthine oxidase. Description: J. Am. Chem. Soc., 2004, 126 (28), pp 8614–8615 DOI: 10.1021/ja0490222 Thu, 01 Jan 2004 00:00:00 GMT http://hdl.handle.net/10362/8724 2004-01-01T00:00:00Z http://hdl.handle.net/10362/8722 Title: Activation of N2O reduction by the fully reduced micro4-sulfide bridged tetranuclear Cu Z cluster in nitrous oxide reductase Authors: Ghosh, Somdatta; Gorelsky, Serge I.; Chen, Peng; Cabrito, Inês; Moura, José J. G.; Moura, Isabel; Solomon, Edward I. Abstract: The tetranuclear CuZ cluster catalyzes the two-electron reduction of N2O to N2 and H2O in the enzyme nitrous oxide reductase. This study shows that the fully reduced 4CuI form of the cluster correlates with the catalytic activity of the enzyme. This is the first demonstration that the S = 1/2 form of CuZ can be further reduced. Complementary DFT calculations support the experimental findings and demonstrate that N2O binding in a bent mu-1,3-bridging mode to the 4CuI form is most efficient due to strong back-bonding from two reduced copper atoms. This back-donation activates N2O for electrophilic attack by a proton. Description: J. Am. Chem. Soc., 2003, 125 (51), pp 15708–15709 DOI: 10.1021/ja038344n Wed, 01 Jan 2003 00:00:00 GMT http://hdl.handle.net/10362/8722 2003-01-01T00:00:00Z http://hdl.handle.net/10362/8721 Title: Incorporation of either molybdenum or tungsten into formate dehydrogenase from Desulfovibrio alaskensis NCIMB 13491; EPR assignment of the proximal iron-sulfur cluster to the pterin cofactor in formate dehydrogenases from sulfate-reducing bacteria Authors: Brondino, Carlos D.; Passeggi, Mario C. G.; Caldeira, Jorge; Almendra, Maria J.; Feio, Maria J.; Moura, José J. G.; Moura, Isabel Abstract: We report the characterization of the molecular properties and EPR studies of a new formate dehydrogenase (FDH) from the sulfate-reducing organism Desulfovibrio alaskensis NCIMB 13491. FDHs are enzymes that catalyze the two-electron oxidation of formate to carbon dioxide in several aerobic and anaerobic organisms. D. alaskensis FDH is a heterodimeric protein with a molecular weight of 126+/-2 kDa composed of two subunits, alpha=93+/-3 kDa and beta=32+/-2 kDa, which contains 6+/-1 Fe/molecule, 0.4+/-0.1 Mo/molecule, 0.3+/-0.1 W/molecule, and 1.3+/-0.1 guanine monophosphate nucleotides. The UV-vis absorption spectrum of D. alaskensis FDH is typical of an iron-sulfur protein with a broad band around 400 nm. Variable-temperature EPR studies performed on reduced samples of D. alaskensis FDH showed the presence of signals associated with the different paramagnetic centers of D. alaskensis FDH. Three rhombic signals having g-values and relaxation behavior characteristic of [4Fe-4S] clusters were observed in the 5-40 K temperature range. Two EPR signals with all the g-values less than two, which accounted for less than 0.1 spin/protein, typical of mononuclear Mo(V) and W(V), respectively, were observed. The signal associated with the W(V) ion has a larger deviation from the free electron g-value, as expected for tungsten in a d(1) configuration, albeit with an unusual relaxation behavior. The EPR parameters of the Mo(V) signal are within the range of values typically found for the slow-type signal observed in several Mo-containing proteins belonging to the xanthine oxidase family of enzymes. Mo(V) resonances are split at temperatures below 50 K by magnetic coupling with one of the Fe/S clusters. The analysis of the inter-center magnetic interaction allowed us to assign the EPR-distinguishable iron-sulfur clusters with those seen in the crystal structure of a homologous enzyme. Description: J Biol Inorg Chem (2004) 9: 145–151 DOI 10.1007/s00775-003-0506-z Thu, 01 Jan 2004 00:00:00 GMT http://hdl.handle.net/10362/8721 2004-01-01T00:00:00Z http://hdl.handle.net/10362/8720 Title: The isolation and characterization of cytochrome c nitrite reductase subunits (NrfA and NrfH) from Desulfovibrio desulfuricans ATCC 27774 Authors: Almeida, Maria Gabriela; Macieira, Sofia; Gonçalves, Luisa L.; Romão, Maria J.; Costa, Cristina; Lampreia, Jorge; Moura, José J. G.; Moura, Isabel; Cunha, Carlos A. Abstract: The cytochrome c nitrite reductase is isolated from the membranes of the sulfate-reducing bacterium Desulfovibrio desulfuricans ATCC 27774 as a heterooligomeric complex composed by two subunits (61 kDa and 19 kDa) containing c-type hemes, encoded by the genes nrfA and nrfH, respectively. The extracted complex has in average a 2NrfA:1NrfH composition. The separation of ccNiR subunits from one another is accomplished by gel filtration chromatography in the presence of SDS. The amino-acid sequence and biochemical subunits characterization show that NrfA contains five hemes and NrfH four hemes. These considerations enabled the revision of a vast amount of existing spectroscopic data on the NrfHA complex that was not originally well interpreted due to the lack of knowledge on the heme content and the oligomeric enzyme status. Based on EPR and Mössbauer parameters and their correlation to structural information recently obtained from X-ray crystallography on the NrfA structure [Cunha, C.A., Macieira, S., Dias, J.M., Almeida, M.G., Gonçalves, L.M.L., Costa, C., Lampreia, J., Huber, R., Moura, J.J.G., Moura, I. & Romão, M. (2003) J. Biol. Chem. 278, 17455-17465], we propose the full assignment of midpoint reduction potentials values to the individual hemes. NrfA contains the high-spin catalytic site (-80 mV) as well as a quite unusual high reduction potential (+150 mV)/low-spin bis-His coordinated heme, considered to be the site where electrons enter. In addition, the reassessment of the spectroscopic data allowed the first partial spectroscopic characterization of the NrfH subunit. The four NrfH hemes are all in a low-spin state (S = 1/2). One of them has a gmax at 3.55, characteristic of bis-histidinyl iron ligands in a noncoplanar arrangement, and has a positive reduction potential. Description: Eur. J. Biochem. 270, 3904–3915 (2003) doi:10.1046/j.1432-1033.2003.03772.x Wed, 01 Jan 2003 00:00:00 GMT http://hdl.handle.net/10362/8720 2003-01-01T00:00:00Z