Crystallization and preliminary X-ray analysis of a nitrate reductase from <i>Desulfovibrio desulfuricans</i> ATCC 27774

Dias, João M.; Bursakov, Sergey A.; Carneiro, C.; Moura, José J. G.; Moura, Isabel; Romão, Maria João

doi:10.1107/s0907444998014735

Cited by 9 publications

(11 citation statements)

References 15 publications

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“…Crystals of these reduced samples were grown at approximately 295 K, with 6% PEG 8K and 100 mM MES, pH 6.0. Single crystals were obtained, which were very similar to the ones obtained aerobically [15]. Initial attempts to flash-cool the crystals in liquid dinitrogen (inside the chamber, in order to maintain the reducing conditions) were, however, unsuccessful.…”

Section: Spectropotentiometric Titrationsupporting

confidence: 49%

“…However, it could not be confirmed by an experiment conducted with 15 N-labeled nitrate because of a strong signal from the dye overlapping that of the Mo(V) ion [9]. With use of the optimized conditions of the spectropotentiometric titration, the turnover signal was developed with 15 Fig. 6 Closeup of the area of the funnel leading to the active site of the perchlorate-bound DdNapA.…”

Section: Structures Of Inhibited Formsmentioning

confidence: 99%

“…In this case it was possible to model either a cyanide ion (with either the nitrogen or the carbon atoms coordinating the molybdenum atom) or a sulfur atom with 75% occupancy giving rise to a better B factor correlation. EPR data taken with labeled cyanide (KC 15 N, I N = 1/2; K 13 CN, I C = 1/2) do not show hyperfine splitting associated with either nitrogen or carbon, which suggests that a cyanide molecule is not coordinated to molybdenum, at least in the EPR-active species [9]. In this context, the results of the refinement for the CYANIDE dataset could be due to a mixture of different forms probably related to the reactivity of the sulfur ligand, suggesting that inhibition by cyanide occurs by cyanolysis of the sulfur ligand in the sixth position in analogy to the cyanolysable sulfido group in xanthine oxidase [31].…”

Section: Structures Of Inhibited Formsmentioning

confidence: 99%

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Periplasmic nitrate reductase revisited: a sulfur atom completes the sixth coordination of the catalytic molybdenum

et al. 2008

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Nitrate reductase from Desulfovibrio desulfuricans ATCC 27774 (DdNapA) is a monomeric protein of 80 kDa harboring a bis(molybdopterin guanine dinucleotide) active site and a [4Fe-4S] cluster. Previous electron paramagnetic resonance (EPR) studies in both catalytic and inhibiting conditions showed that the molybdenum center has high coordination flexibility when reacted with reducing agents, substrates or inhibitors. As-prepared DdNapA samples, as well as those reacted with substrates and inhibitors, were crystallized and the corresponding structures were solved at resolutions ranging from 1.99 to 2.45 Å . The good quality of the diffraction data allowed us to perform a detailed structural study of the active site and, on that basis, the sixth molybdenum ligand, originally proposed to be an OH/OH 2 ligand, was assigned as a sulfur atom after refinement and analysis of the B factors of all the structures. This unexpected result was confirmed by a single-wavelength anomalous diffraction experiment below the iron edge (k = 1.77 Å ) of the as-purified enzyme. Furthermore, for six of the seven datasets, the S-S distance between the sulfur ligand and the Sc atom of the molybdenum ligand Cys A140 was substantially shorter than the van der Waals contact distance and varies between 2.2 and 2.85 Å , indicating a partial disulfide bond. Preliminary EPR studies under catalytic conditions showed an EPR signal designated as a turnover signal (g values 1.999, 1.990, 1.982) showing hyperfine structure originating from a nucleus of unknown nature. Spectropotentiometric studies show that reduced methyl viologen, the electron donor used in the catalytic reaction, does not interact directly with the redox cofactors. The turnover signal can be obtained only in the presence of the reaction substrates. With use of the optimized conditions determined by spectropotentiometric titration, the turnover signal was developed with 15 N-labeled nitrate and in D 2 O-exchanged DdNapA samples. These studies indicate that this signal is not associated with a Mo(V)-nitrate adduct and that the hyperfine structure originates from two equivalent solvent-exchangeable protons. The new coordination sphere of molybdenum proposed on the basis of our studies led us to revise the currently accepted reaction mechanism for periplasmic nitrate reductases. Proposals for a new mechanism are discussed taking into account a molybdenum and ligandbased redox chemistry, rather than the currently accepted redox chemistry based solely on the molybdenum atom.

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Section: Spectropotentiometric Titrationsupporting

confidence: 49%

Section: Structures Of Inhibited Formsmentioning

confidence: 99%

Section: Structures Of Inhibited Formsmentioning

confidence: 99%

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Periplasmic nitrate reductase revisited: a sulfur atom completes the sixth coordination of the catalytic molybdenum

et al. 2008

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“…5) [44,45]. Dd Nap is a monomeric protein of ellipsoidal shape with an a/b type fold organized in four domains, all involved in cofactor binding.…”

Section: Molecular and Spectroscopic Propertiesmentioning

confidence: 99%

Bacterial nitrate reductases: Molecular and biological aspects of nitrate reduction

González

Correia

Moura

et al. 2006

Journal of Inorganic Biochemistry

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226

174

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“…The enzymology and bioenergetics of respiratory nitrite ammonifi cation have been recently reviewed [Simon, 2002]. In respiratory nitrite ammonification, nitrite is reduced to ammonia without the release of intermediate products, such as NO or N 2 O, in a sixelectron step catalyzed by a ccNiR, the so-called NrfA protein Bamford et al, 2002;Cunha et al, 2003;Dias et al, 2000;Einsle et al, 1999Einsle et al, , 2000. This process can be regarded as a short circuit that bypasses denitrifi cation and nitrogen fi xation [Rudolf and Kroneck, 2005].…”

Section: Reduction Of Nitrite To Ammonia (Dissimilatory Nitrate Ammonmentioning

confidence: 99%

Key Bacterial Multi-Centered Metal Enzymes Involved in Nitrate and Sulfate Respiration

et al. 2005

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Many essential life processes, such as photosynthesis, respiration, nitrogen fixation, depend on transition metal ions and their ability to catalyze multi-electron redox and hydrolytic transformations. Here we review some recent structural studies on three multi-site metal enzymes involved in respiratory processes which represent important branches within the global cycles of nitrogen and sulfur: (i) the multi-heme enzyme cytochrome c nitrite reductase, (ii) the FAD, FeS-enzyme adenosine-5′-phosphosulfate reductase, and (iii) the siroheme, FeS-enzyme sulfite reductase. Structural information comes from X-ray crystallography and spectroscopical techniques, in special cases catalytically competent intermediates could be trapped and characterized by X-ray crystallography.

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Crystallization and preliminary X-ray analysis of a nitrate reductase from Desulfovibrio desulfuricans ATCC 27774

Cited by 9 publications

References 15 publications

Periplasmic nitrate reductase revisited: a sulfur atom completes the sixth coordination of the catalytic molybdenum

Periplasmic nitrate reductase revisited: a sulfur atom completes the sixth coordination of the catalytic molybdenum

Bacterial nitrate reductases: Molecular and biological aspects of nitrate reduction

Key Bacterial Multi-Centered Metal Enzymes Involved in Nitrate and Sulfate Respiration

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