Conformation change of cytochrome c: II. Ferricytochrome c refinement at 1·8 Å and comparison with the ferrocytochrome structure

Abstract

Tuna ferricytochrome c has been crystallographically refined at a resolution of 1·8 Å using Diamond real-space methods followed by Jack Levitt restrained energy and reciprocal space refinement, with Fourier and difference Fourier map monitoring. The final R factor for two independent cytochromes plus 49 solvent molecules, using 16,831 reflections to 1.8 Å is 20·8%. The structure is essentially the same as described at 2.0 Å (Swanson et al., 1977), but the increased accuracy now permits detailed comparison with the reduced cytochrome c molecule (preceding paper). Twelve water molecules are found in identical positions in both oxidized molecules and also in the reduced. Three of these are buried: one between Asn52, Tyr67 and Thr78 to the lower left of the haem, a second inside the 20s loop, and a third below the buried haem propionate. The first of these three buried water molecules is involved in a concerted shift of side-chains and even main-chain at the bottom of the molecule, that accompanies the change in redox state of the haem. The shift of this water molecule and a slight outward movement of the haem both give the haem a more hydrophilic environment in ferricytochrome c. This same buried water molecule may be involved in the alkaline conformation transition that ferricytochrome c undergoes at pH 9·4, assisting in the replacement of Met80 by a different low-spin ligand. The absence of such transition in ferrocytochrome c until pH 12 is reached may be ascribed to the shift in water position observed in the reduced state. The water molecule buried in the 20s loop, in turn, may be involved in the acid transformation that ferricytochrome c undergoes at pH 2·5. The conformations observed by X-rays for both ferricytochrome c and ferrocytochrome c appear to be post-transfer states, i.e. states that are most compatible with the particular redox state of the haem, rather than being poised to give up or receive electrons. These conformations have been interpreted as being effects of changes in haem redox state rather than causes, but if binding to another macromolecule were to induce either conformation, then transfer of an electron might thereby be facilitated.