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A combination of structural, functional, and mutagenic experiments has been used
to study the roles of the invariant Phe82 and highly conserved Leu85 residues in
cytochrome c, especially with respect to the complexation interface with
electron transfer partners and maintenance of the hydrophobic heme pocket.
Structural analyses show that the F82Y, L85A, and F82Y/L85A mutant proteins all
retain the characteristic cytochrome c fold, but that conformational alterations
are introduced in the direct vicinity of the mutation sites. In particular, the
additional hydroxyl group of Tyr82 is in direct spatial conflict with the side
chain of Leu85 in the F82Y mutant protein, leading to rotation of the side chain
of Tyr82 out toward the protein surface. This strain is relieved in the
F82Y/L85A mutant protein where the phenyl ring of Tyr82 is accommodated in a
conformation comparable to that of the phenylalanine normally present at this
location. In addition, the available space vacated by the replacement of Leu85
with an alanine allows for the inclusion of two new internal water molecules,
one of which is bound to Tyr82 and the other to Arg13. In contrast, in the L85A
mutant protein, no internal water molecules are observed in this exclusively
hydrophobic pocket, which is partially filled by shifts in nearby side chains.
Overall, the conformational changes observed result from the optimization of
side chain packing to reflect the spatial requirements of new side chains, the
minimization of both vacant internal space and the solvent exposure of
hydrophobic groups, and the attainment of maximal hydrogen bonding between
available polar groups.(ABSTRACT TRUNCATED AT 250 WORDS)
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