Crystallographic determination of the heme orientation and location of the cyanide binding site in yeast cytochrome c peroxidase.

The location and orientation of the heme in yeast cytochrome c peroxidase has been determined from a 2.5 A electron density map phased with two isomorphous mercury derivatives. The noncovalently bound heme is located in a crevice open to the solvent and is oriented so that the two propionate side chains extend toward the surface of the enzyme molecule. The crevice is lined with side chains that apparently are aromatic. A channel, formed in part by one of these side chains and by pyrrole IV of the heme, connects the sixth coordination position to the surface. Presumably this channel provides access to the sixth coordination position for hydroperoxide substrates. In agreement with solution studies by other workers, we find that the fifth axial heme ligand is NE2 of a histidine side chain and that the sixth is a solvent molecule. Crystals soaked in solutions containing KCN are isomorphous with parent crystals but have a distinctly different color. A 2.6 A difference Fourier computed with [F(cyanide) F(parent)] coefficients and multiple isomorphous replacement phases showed significant difference density only within the heme crevice. Interpretation of the difference map revealed that when cyanide binds to yeast cytochrome c peroxidase: 1) the sixth axial ligand is displaced by cyanide; 2) the amino acid side chain that forms part of the peroxide access channel moves about 0.4 .& away from the heme and a second aromatic residue lying about 3.5 A above pyrrole II moves a few tenths of an angstrom away from the sixth coordination position; and 3) the heme itself is perturbed.