High-resolution three-dimensional structure of horse heart cytochrome c

Abstract

The 1.94A˚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 β-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.