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High-resolution three-dimensional structural analyses of yeast iso-1-cytochrome
c have now been completed in both oxidation states using isomorphous crystalline
material and similar structure determination methodologies. This approach has
allowed a comprehensive comparison to be made between these structures and the
elucidation of the subtle conformational changes occurring between oxidation
states. The structure solution of reduced yeast iso-1-cytochrome c has been
published and the determination of the oxidized protein and a comparison of
these structures are reported herein. Our data show that oxidation
state-dependent changes are expressed for the most part in terms of adjustments
to heme structure, movement of internally bound water molecules and segmental
thermal parameter changes along the polypeptide chain, rather than as explicit
polypeptide chain positional shifts, which are found to be minimal. This result
is emphasized by the retention of all main-chain to main-chain hydrogen bond
interactions in both oxidation states. Observed thermal factor changes primarily
affect four segments of polypeptide chain. Residues 37-39 show less mobility in
the oxidized state, with Arg38 and its side-chain being most affected. In
contrast, residues 47-59, 65-72 and 81-85 have significantly higher thermal
factors, with maximal increases being observed for Asn52, Tyr67 and Phe82. The
side-chains of two of these residues are hydrogen bonded to the internally bound
water molecule, Wat166, which shows a large 1.7 A displacement towards the
positively charged heme iron atom in the oxidized protein. Further analyses
suggest that Wat166 is a major factor in stabilizing both oxidation states of
the heme through differential orientation of dipole moment, shift in distance to
the heme iron atom and alterations in the surrounding hydrogen bonding network.
It also seems likely that Wat166 movement leads to the disruption of the
hydrogen bond from the side-chain of Tyr67 to the Met80 heme ligand, thereby
further stabilizing the positively charged heme iron atom in oxidized cytochrome
c. In total, there appear to be three regions about which oxidation
state-dependent structural changes are focussed. These include the pyrrole ring
A propionate group, Wat166 and the Met80 heme ligand. All three of these foci
are linked together by a network of intermediary interactions and are localized
to the Met80 ligand side of the heme group. Associated with each is a
corresponding nearby segment of polypeptide chain having a substantially higher
mobility in the oxidized protein.(ABSTRACT TRUNCATED AT 400 WORDS)
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