Crystal structure of nitric oxide inhibited cytochrome c peroxidase

Abstract

We have collected X-ray diffraction data from a crystal of cytochrome c peroxidase (CCP) complexed with inhibitor nitric oxide to a resolution of 2.55 .ANG.. A difference Fourier map shows density indicating the NO ligand is bound to the heme iron at the sixth corrdination site in a bent configuration. Structural adjustments were determined by least-squares refinement that yielded an agreement residual of R = 0.18. The orientation of the ligand, tilting toward Arg-48, causes adjustment in the position of this nearby polar side chain. As a model for the substrate hydrogen peroxide, this geometry is consistent with the suggestion that Arg-48 serves to polarize the O-O peroxide bond to promote heterolytic cleavage of the bond [Poulos, T. L., .ALPHA.MP Kraut, J. (1980) J. Biol. Chem. 255, 8199-8205]. Strong difference density is also observed near residues 190-194, especially around the indole ring of Trp-191. The density indicates movement of the indole ring away from the proximal His-175 imidazole ring by about 0.25 .ANG., which appears to cause perturbation of the neighboring residues. The response of Trp-191 on the proximal side of the heme to binding nitric oxide on the distal side probably results from delocalization of the electron density of the ligand. Relevant to this is the recent finding that a mutant in which Trp-191 is replaced by phenylalanine has dramatically reduced activity, less than 0.05% of the parent activity [Mauro. J. M. Fishel. L. A., hazzard, J. T., Meyer, T. E., Tollin, G., Cusanovich, M. A., and Kraut, J. (1988) Biochemistry 27, 6243-6256]. Characterization of this mutant showed in particular that electron transfer from cytochrome c was severely hindred. This mutagenesis results combined with the sensitivity of the position of Trp-191 to the electronic character of the sixth coordination site ligand leads us to speculate on the role of Trp-191 in electron transfer.