| Abstract
| - This article reports the first X-ray structure of the soluble form of a c-type cytochrome isolatedfrom a Gram-positive bacterium. Bacillus pasteurii cytochrome c553, characterized by a low reductionpotential and by a low sequence homology with cytochromes from Gram-negative bacteria or eukaryotes,is a useful case study for understanding the structure−function relationships for this class of electron-transfer proteins. Diffraction data on a single crystal of cytochrome c553 were obtained using synchrotronradiation at 100 K. The structure was determined at 0.97-Å resolution using ab initio phasing andindependently at 1.70 Å in an MAD experiment. In both experiments, the structure solution exploited thepresence of a single Fe atom as anomalous scatterer in the protein. For the 0.97-Å data, the phasing wasbased on a single data set. This is the most precise structure of a heme protein to date. The crystallizedcytochrome c553 contains only 71 of the 92 residues expected from the intact protein sequence, lackingthe first 21 amino acids at the N-terminus. This feature is consistent with previous evidence that this tail,responsible for anchoring the protein to the cytoplasm membrane, is easily cleaved off during the purificationprocedure. The heme prosthetic group in B. pasteurii cytochrome c553 is surrounded by three α-helices ina compact arrangement. The largely exposed c-type heme group features a His−Met axial coordinationof the Fe(III) ion. The protein is characterized by a very asymmetric charge distribution, with the exposedheme edge located on a surface patch devoid of net charges. A structural search of a representative set ofprotein structures reveals that B. pasteurii cytochrome c553 is most similar to Pseudomonas cytochromesc551, followed by cytochromes c6, Desulfovibrio cytochrome c553, cytochromes c552 from thermophiles,and cytochromes c from eukaryotes. Notwithstanding a low sequence homology, a structure-based alignmentof these cytochromes shows conservation of three helical regions, with different additional secondarystructure motifs characterizing each protein. In B. pasteurii cytochrome c553, these motifs are representedby the shortest interhelix connecting fragments observed for this group of proteins. The possible relationshipsbetween heme solvent accessibility and the electrochemical reduction potential are discussed.
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