Primary Structure Characterization of a Rhodocyclus tenuis Diheme Cytochrome c Reveals the Existence of Two Different Classes of Low-Potential Diheme Cytochromes c in Purple Phototropic Bacteria Bart Devreese,* Ann Brige ´,* Katrien Backers,* Gonzalez Van Driessche,* Terrance E. Meyer,† Michael A. Cusanovich,† and Jozef J. Van Beeumen* ,1 *Department of Biochemistry, Physiology, and Microbiology, Laboratory of Protein Biochemistry and Protein Engineering, University of Gent, Ledeganckstraat 35, 9000 Gent, Belgium; and †Department of Biochemistry, University of Arizona, Tucson, Arizona 85721 Received December 2, 1999, and in revised form June 19, 2000 The complete amino acid sequence of a 26-kDa low redox potential cytochrome c-551 from Rhodocyclus tenuis was determined by a combination of Edman degradation and mass spectrometry. There are 240 residues including two heme binding sites at positions 41, 44, 128, and 132. There is no evidence for gene doubling. The only known homolog of Rc. tenuis cyto- chrome c-551 is the diheme cytochrome c-552 from Pseudomonas stutzeri which contains 268 residues and heme binding sites at nearly identical positions. There is 44% overall identity between the Rc. tenuis and Ps. stutzeri cytochromes with 10 internal inser- tions and deletions. The Ps. stutzeri cytochrome is part of a denitrification gene cluster, whereas Rc. tenuis is incapable of denitrification, suggesting dif- ferent functional roles for the cytochromes. Histidines at positions 45 and 133 are the fifth heme ligands and conserved histidines at positions 29, 209, and 218 and conserved methionines at positions 114 and 139 are potential sixth heme ligands. There is no obvious ho- mology to the low-potential diheme cytochromes char- acterized from other purple bacterial species such as Rhodobacter sphaeroides. There are therefore at least two classes of low-potential diheme cytochromes c found in phototrophic bacteria. There is no more than 11% helical secondary structure in Rc. tenuis cyto- chrome c-551 suggesting that there is no relationship to class I or class II c-type cytochromes. © 2000 Academic Press Key Words: amino acid sequence; diheme; Rhodocy- clus tenuis; low-potential cytochrome. Low-redox-potential cytochromes have been re- ported in a number of purple phototrophic bacteria, although they have not been found in all species exam- ined (1, 2). These proteins have not received as much attention as have high-potential cytochromes and no sequences had been reported until recently. The low- potential cytochromes from Rhodobacter sphaeroides and Rhodobacter adriaticus have two heme-binding sites per 126 and 139 residues, respectively (3). These proteins have only two homologs, translated gene SLL1886 from Synechocystis PCC6803, which contains two potential heme binding sites per 188 residues (4), and a diheme cytochrome from the metal-oxide-respir- ing bacterium Shewanella putrefaciens (www.tigr.org), which has two hemes per 149 residues. Adjacent to the low-potential diheme cytochrome gene in Shewanella is the gene for a homolog of another Rb. sphaeroides protein, the oxygen binding cytochrome called SHP. These genes form an operon and the proteins are likely to form an electron transfer pathway leading to the reduction of oxygen. We have shown in an unpublished work (K. Backers et al.) that a similar three-gene operon exists in Rb. sphaeroides. To determine the distribution of this pathway in purple bacteria, we initiated sequence studies of the Rhodocyclus tenuis diheme cytochrome c -551 with the eventual goal to clone and overexpress it for structural determination. However, we find that the Rc. tenuis sequence shows 1 To whom correspondence should be addressed at Vakgroep Bio- chemie, Fysiologie en Mikrobiologie, Laboratorium voor Eiwitbioche- mie en Eiwitengineering, Ledeganckstraat 35, B-9000 Gent, Bel- gium. Fax: 32-9-264 5338. E-mail: jozef.vanbeeumen@rug.ac.be. 0003-9861/00 $35.00 53 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved. Archives of Biochemistry and Biophysics Vol. 381, No. 1, September 1, pp. 53– 60, 2000 doi:10.1006/abbi.2000.1971, available online at http://www.idealibrary.com on