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PDBsum entry 2v3v
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Oxidoreductase
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PDB id
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2v3v
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References listed in PDB file
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Key reference
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Title
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Periplasmic nitrate reductase revisited: a sulfur atom completes the sixth coordination of the catalytic molybdenum.
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Authors
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S.Najmudin,
P.J.González,
J.Trincão,
C.Coelho,
A.Mukhopadhyay,
N.M.Cerqueira,
C.C.Romão,
I.Moura,
J.J.Moura,
C.D.Brondino,
M.J.Romão.
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Ref.
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J Biol Inorg Chem, 2008,
13,
737-753.
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PubMed id
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Abstract
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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 A. 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 (lambda = 1.77 A) of the as-purified enzyme.
Furthermore, for six of the seven datasets, the S-S distance between the sulfur
ligand and the Sgamma 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
A, 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 ligand-based redox chemistry, rather than
the currently accepted redox chemistry based solely on the molybdenum atom.
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Secondary reference #1
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Title
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Epr and redox properties of periplasmic nitrate reductase from desulfovibrio desulfuricans atcc 27774.
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Authors
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P.J.González,
M.G.Rivas,
C.D.Brondino,
S.A.Bursakov,
I.Moura,
J.J.Moura.
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Ref.
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J Biol Inorg Chem, 2006,
11,
609-616.
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PubMed id
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