CRYSTAL-STRUCTURE OF THE DIHEME CYTOCHROME C(4) FROM PSEUDOMONAS-STUTZERI DETERMINED AT 2.2-ANGSTROM RESOLUTION

Background: Cytochromes c(4) are dihaem cytochromes c found in a varie ty of bacteria. They are assumed to take part in the electron-transpor t systems associated with both aerobic and anaerobic respiration. The cytochrome c(4) proteins are located in the periplasm, predominantly b ound to the inner membrane, and are able to transfer electrons between membrane-bound reduction systems and terminal oxidases. Alignment of cytochrome c(4) sequences from three bacteria, Pseudomonas aeruginosa, Pseudomonas slutzeri and Azotobacter vinelandii, suggests that these dihaem proteins are composed of two similar domains. Two distinctly di fferent redox potentials have been measured for the Ps. stutzeri cytoc hrome c(4), however. Results: The crystal structure of the dihaem cyto chrome c(4) from Ps. slutzeri has been determined to 2.2 Angstrom reso lution by isomorphous replacement. The model, consisting of two entire cytochrome c(4) molecules and 138 water molecules in the asymmetric u nit, was refined to an R value of 20.1% for all observations in the re solution range 8-2.2 Angstrom. The molecule is organized in two cytoch rome c-like domains that are related by a pseudo-twofold axis, The sym metry is virtually perfectly close to the twofold axis, which passes t hrough a short hydrogen bond between the two haem propionic acid group s, connecting the redox centre of each domain. This haem-haem interact ion is further stabilized by an extensive symmetrical hydrogen-bond ne twork. The twofold symmetry is not present further away from the axis, however, and the cytochrome c(4) molecule can be considered to be a d ipole with charged residues unevenly distributed between the two domai ns. The haem environments in the two domains show pronounced differenc es, mainly on the methionine side of the haem group. Conclusions: The structure, in conjunction with sequence alignment, suggests that the c ytochrome protein has evolved by duplication of a cytochrome c gene. T he difference in charge distribution around each haem group in the two domains allows the haem group in the N-terminal domain to be associat ed with the lower redox potential of 241 mV and the C-terminal haem gr oup with the higher potential of 328 mV, The molecular dipole characte ristic of cytochrome c(4) is important for its interaction with, and r ecognition of, its redox partners. In cytochrome c(4), the hydrogen-bo nd network (between residues that are conserved in all known cytochrom e c(4) subspecies) seems to provide an efficient pathway for an intram olecular electron transfer that can ensure cooperativity between the t wo redox centres. The C-pyrrole corners of the haem edges are potentia l sites for external electron exchange.