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Title
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The role of a conserved internal water molecule and its associated hydrogen bond network in cytochrome c.
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Authors
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A.M.Berghuis,
J.G.Guillemette,
G.McLendon,
F.Sherman,
M.Smith,
G.D.Brayer.
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Ref.
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J Mol Biol, 1994,
236,
786-799.
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PubMed id
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Abstract
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High resolution three-dimensional structures for the N52I and N52I-Y67F yeast
iso-1-cytochrome c variants have been completed in both oxidation states. The
most prominent structural difference observed in both mutant proteins is the
displacement of a conserved, internally bound water molecule (Wat166) from the
protein matrix. In wild-type yeast iso-1-cytochrome c the position and
orientation of this water molecule is found to be dependent on the oxidation
state of the heme iron atom. Overall our results suggest the function of Wat166
and its associated hydrogen bond network is threefold. First, the presence of
Wat166 provides a convenient mechanism to modify the hydrogen bond network
involving several residues near the Met80 ligand in an oxidation state dependent
manner. Second, Wat166 is necessary for the maintenance of the spatial
relationships between nearby side-chains and the hydrogen bond interactions
formed between these groups in this region of the protein. An essential part of
this role is ensuring the proper conformation of the side-chain of Tyr67 so that
it forms a hydrogen bond interaction with the heme ligand Met80. This hydrogen
bond influences the electron withdrawing power of the Met80 ligand and is
therefore a factor in controlling the midpoint reduction potential of cytochrome
c. Elimination of this interaction in the N52I-Y67F mutant protein or
elimination of Wat166 in the N52I protein with the subsequent disruption in the
position and interactions of the Tyr67 side-chain, leads to a drop of
approximately 56 mV in the observed midpoint reduction potential of the heme
group. Third, Wat166 also appears to mediate increases in the mobility of three
nearby segments of polypeptide chain when cytochrome c is in the oxidized state.
Previous studies have proposed these changes may be related to oxidation state
dependent interactions between cytochrome c and its redox partners. Coincident
with the absence of Wat166, such mobility changes are not observed in the N52I
and N52I-Y67F mutant proteins. It is possible that much of the increased protein
stability observed for both mutant proteins may be due to this factor. Finally,
our results show that neither heme iron charge nor heme plane distortion are
responsible for oxidation state dependent conformational changes in the pyrrole
A propionate region. Instead, the changes observed appear to be driven by the
change in conformation that the side-chain of Asn52 experiences as the result of
oxidation state dependent movement of Wat166.
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