MAD structure of Pseudomonas nautica dimeric cytochrome c(552) mimicks thec(4) dihemic cytochrome domain association

The monohemic cytochrome c(552) from Pseudomonas nautica (c(552)-Pn) is tho ught to be the electron donor to cytochrome cd(1), the so-called nitrite re ductase (NiR). It shows as high levels of activity and affinity for the P. nautica NiR (NiR-Pn), as the Pseudomonas aeruginosa enzyme (NiR-Pa). Since cytochrome c(552) is by far the most abundant electron carrier in the perip lasm, it is probably involved in numerous other reactions. Its sequence is related to that of the c type cytochromes, but resembles that of the dihemi c c(4) cytochromes even more closely. The three-dimensional structure of P. nautica cytochrome c(552) has been so lved to 2.2 Angstrom resolution using the multiple wavelength anomalous dis persion (MAD) technique, taking advantage of the presence of the eight Fe h eme ions in the asymmetric unit. Density modification procedures involving 4-fold non-crystallographic averaging yielded a model with an R-factor valu e of 17.8% (R-free = 20.8%). Cytochrome c(552) forms a tight dimer in the c rystal, and the dimer interface area amounts to 19% of the total cytochrome surface area. Four tighly packed dimers form the eight molecules of the as ymmetric unit. The c(552) dimer is superimposable on each domain of the monomeric cytochro me c(4) from Pseudomonas stutzeri (c(4)-Ps), a dihemic cytochrome, and on t he dihemic c domain of flavocytochrome c of Chromatium vinosum (Fcd-Cv). Th e interacting residues which form the dimer are both similar in character a nd position, which is also true for the propionates. The dimer observed in the crystal also exists in solution. It has been hypothesised that the dihe mic c(4)-Ps may have evolved via monohemic cytochrome c gene duplication fo llowed by evolutionary divergence and the adjunction of a connecting linker . Ln this process, our dimeric c(552) structure might be said to constitute a "living fossile" occurring in the course of evolution between the format ion of the dimer and the gene duplication and fusion. The availability of t he structure of the cytochrome c(552)-Pn and that of NiR from P. aeruginosa made it possible to identify putative surface patches at which the docking of c(552) to NiR-Pn may occur. (C) 1999 Academic Press.