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The 1.94 A resolution three-dimensional structure of oxidized horse heart
cytochrome c has been elucidated and refined to a final R-factor of 0.17. This
has allowed for a detailed assessment of the structural features of this
protein, including the presence of secondary structure, hydrogen-bonding
patterns and heme geometry. A comprehensive analysis of the structural
differences between horse heart cytochrome c and those other eukaryotic
cytochromes c for which high-resolution structures are available (yeast iso-1,
tuna, rice) has also been completed. Significant conformational differences
between these proteins occur in three regions and primarily involve residues 22
to 27, 41 to 43 and 56 to 57. The first of these variable regions is part of a
surface beta-loop, whilst the latter two are located together adjacent to the
heme group. This study also demonstrates that, in horse cytochrome c, the
side-chain of Phe82 is positioned in a co-planar fashion next to the heme in a
conformation comparable to that found in other cytochromes c. The positioning of
this residue does not therefore appear to be oxidation-state-dependent. In
total, five water molecules occupy conserved positions in the structures of
horse heart, yeast iso-1, tuna and rice cytochromes c. Three of these are on the
surface of the protein, serving to stabilize local polypeptide chain
conformations. The remaining two are internally located. One of these mediates a
charged interaction between the invariant residue Arg38 and a nearby heme
propionate. The other is more centrally buried near the heme iron atom and is
hydrogen bonded to the conserved residues Asn52, Tyr67 and Thr78. It is shown
that this latter water molecule shifts in a consistent manner upon change in
oxidation state if cytochrome c structures from various sources are compared.
The conservation of this structural feature and its close proximity to the heme
iron atom strongly implicate this internal water molecule as having a functional
role in the mechanism of action of cytochrome c.
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