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PDBsum entry 2flq
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Oxidoreductase
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PDB id
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2flq
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References listed in PDB file
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Key reference
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Title
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Structure and reactivity of a thermostable prokaryotic nitric-Oxide synthase that forms a long-Lived oxy-Heme complex.
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Authors
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J.Sudhamsu,
B.R.Crane.
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Ref.
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J Biol Chem, 2006,
281,
9623-9632.
[DOI no: ]
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PubMed id
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Abstract
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In an effort to generate more stable reaction intermediates involved in
substrate oxidation by nitric-oxide synthases (NOSs), we have cloned, expressed,
and characterized a thermostable NOS homolog from the thermophilic bacterium
Geobacillus stearothermophilus (gsNOS). As expected, gsNOS forms nitric oxide
(NO) from l-arginine via the stable intermediate N-hydroxy l-arginine (NOHA).
The addition of oxygen to ferrous gsNOS results in long-lived heme-oxy complexes
in the presence (Soret peak 427 nm) and absence (Soret peak 413 nm) of
substrates l-arginine and NOHA. The substrate-induced red shift correlates with
hydrogen bonding between substrate and heme-bound oxygen resulting in conversion
to a ferric heme-superoxy species. In single turnover experiments with NOHA, NO
forms only in the presence of H(4)B. The crystal structure of gsNOS at 3.2 AA of
resolution reveals great similarity to other known bacterial NOS structures,
with the exception of differences in the distal heme pocket, close to the oxygen
binding site. In particular, a Lys-356 (Bacillus subtilis NOS) to Arg-365
(gsNOS) substitution alters the conformation of a conserved Asp carboxylate,
resulting in movement of an Ile residue toward the heme. Thus, a more
constrained heme pocket may slow ligand dissociation and increase the lifetime
of heme-bound oxygen to seconds at 4 degrees C. Similarly, the ferric-heme NO
complex is also stabilized in gsNOS. The slow kinetics of gsNOS offer promise
for studying downstream intermediates involved in substrate oxidation.
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Figure 1.
FIGURE 1. Current mechanistic model for NO biosynthesis by
NOS adapted from Stuehr et al. (18). Formation of citrulline,
NO, and ferric heme marks the end of one catalytic cycle. In
some mammalian NOS isozymes, further reduction of the
ferric-heme nitrosyl complex competes with NO release from the
active center (18).
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Figure 9.
FIGURE 9. Comparison of the active sites of bsNOS (orange)
and gsNOS (yellow). In bsNOS, Lys-356 does not interact with
Asp-216. A Lys to Arg substitution in gsNOS allows Arg-365 to
hydrogen bond with Asp-225 (3.2 Å), altering its side
chain position. This change in structure appears to be
correlated with movement of Ser-224 that in turn pushes Ile-223
into the active site, reducing the distance between the  -carbon
of Ile-223 and the heme iron atom from 6.7 Å (bsNOS) to
6.1 Å (gsNOS).
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2006,
281,
9623-9632)
copyright 2006.
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