Characterization of DorC from Rhodobacter capsulatus, a c-type Cytochrome Involved in Electron Transfer to Dimethyl Sulfoxide Reductase

Abstract

The dorC gene of the dimethyl sulfoxide respiratory (dor) operon of Rhodobacter capsulatus encodes a pentaheme c-type cytochrome that is involved in electron transfer from ubiquinol to periplasmic dimethyl sulfoxide reductase. DorC was expressed as a C-terminal fusion to an 8-amino acid FLAG epitope and was purified from detergent-solubilized membranes by ion exchange chromatography and immunoaffinity chromatography. The DorC protein had a subunit M r ‫؍‬ 46,000, and pyridine hemochrome analysis indicated that it contained 5 mol heme c/mol DorC polypeptide, as predicted from the derived amino acid sequence of the dorC gene. The reduced form of DorC exhibited visible absorption maxima at 551.5 nm (␣-band), 522 nm (␤-band), and 419 nm (Soret band). Redox potentiometry of the heme centers of DorC identified five components (n ‫؍‬ 1) with midpoint potentials of ؊34, ؊128, ؊184, ؊185, and ؊276 mV. Despite the low redox potentials of the heme centers, DorC was reduced by duroquinol and was oxidized by dimethyl sulfoxide reductase.The ability to use Me 2 SO and trimethylamine-N-oxide (TMAO) 1 as electron acceptors is widespread among facultative aerobic bacteria and the organization of the Me 2 SO and TMAO respiratory chains is now well defined (1). In the Me 2 SO respiratory system of purple photosynthetic bacteria such as Rhodobacter capsulatus, electrons are transferred from primary dehydrogenases via the ubiquinone pool to a periplasmic Me 2 SO reductase (2). Recent sequence and mutational analysis of the Me 2 SO respiratory gene cluster from photosynthetic bacteria has identified a pentaheme c-type cytochrome (DorC) as the likely mediator of electron transfer from ubiquinol to Me 2 SO reductase (3).2 The TMAO respiratory (Tor) system of Escherichia coli (5) is very similar to the Dor system of R. capsulatus, and they differ from the Me 2 SO respiratory (Dms) system of E. coli. The Me 2 SO reductase (DmsABC) of E. coli can be purified as a menaquinol-oxidizing Me 2 SO reductase complex that lacks c-type cytochromes (6). Me 2 SO reductase from R. capsulatus has been purified as a monomeric protein containing a pterin molybdenum cofactor (Moco) as its only prosthetic group (7). This property of Me 2 SO reductase has many advantages for spectroscopic characterization of Moco, but a major deficiency has been the lack of a physiologically relevant electron donor. To overcome this problem we describe in this paper the purification and characterization of DorC.The derived amino acid sequence of Rhodobacter DorC indicated that it was related to members of the NirT class of tetraheme c-type cytochromes (3).2 However, DorC is predicted to be a pentaheme cytochrome with a fifth c-type heme binding motif in the C-terminal polypeptide, which is absent from the tetraheme members of the NirT class. Almost all of the members of the NirT class are involved in an anaerobic respiratory pathway, and their role appears to be to catalyze electron transfer from the Q-pool to a periplasmic terminal reductase (8). Although molecular geneti...

Electron Transfer and Binding of the c-Type Cytochrome TorC to the Trimethylamine N-Oxide Reductase in Escherichia coli

“…The midpoint redox potential described by Shaw et al (21) for purified DorC, a TorC pentahemic homologue, is in the same range of values (Ϫ276, Ϫ185, Ϫ184, Ϫ128 mV, and Ϫ34 mV) as those detailed above for NapC. Surprisingly, all the potentials exhibited by DorC are negative, while a positive one has been detected for TorC.…”

“…The N-terminal region of TorC, which is homologous to NirT and NapC, possesses the four heme binding sites, whereas the C-terminal part, which is present specifically in the Me 2 SO/TMAO respiratory systems, carries the fifth heme motif (5). Recently, two members of the NirT/NapC family, NapC and the TorC homologue DorC, have been characterized, and the redox potential values determined for the four hemes of NapC and the five hemes of DorC were all negative (20,21).…”

Bacterial respiration: a flexible process for a changing environment 1999 Fleming Lecture (Delivered at the 144th meeting of the Society for General Microbiology, 8 September 1999)

“…This is true for a number of bacterial respiratory systems that involve periplasmic oxido-reductases and one solution to the problem seems to have been the evolution of the NapC family of membrane-anchored tetra\penta-haem cytochromes (Roldan et al, 1998). Members of this family have been implicated in mediating electron transfer from quinols to a range of oxido-reductases that include the nitrate reductase (NapC) (Roldan et al, 1998), the periplasmic DMSO reductase (DorC) (Shaw et al, 1999), the TMAO reductase (TorC) and some cytochrome cd " nitrite reductases (NirT) (Jungst et al, 1991). In the case of CycB (Bergmann et al, 1994), they may also serve to transfer electrons into the ubiquinone (UQ) pool during nitrification by Nitrosomonas europaea (Fig.…”

During Cytochrome c Maturation CcmI Chaperones the Class I Apocytochromes until the Formation of Their b-Type Cytochrome Intermediates

“…These include the class I C-terminally membrane-bound cytochrome c 1 subunit of the cytochrome bc 1 complex (8) and the N-terminally membrane-attached cytochrome c p and c o subunits of the cbb 3 -type oxygen reductase (9,10), as well as the soluble cytochrome c 2 and the N-terminally membrane-attached cytochrome c y as electron carriers (11,12). The class II soluble high-spin cytochrome cЈ is involved in NO detoxification (13), and the class III membrane-attached pentaheme c-type cytochrome DorC con-veys electrons from the Q/QH 2 pool to dimethyl sulfoxide (DMSO) 3 reducing it to dimethylsulfide (14). R. capsulatus and other ␣-and ␥-proteobacteria, archaea, and mitochondria of plants and red algae carry out the process of covalent heme ligation to the c-type apocytochromes via a membrane complex, designated as cytochrome c maturation (Ccm) System I (15)(16)(17)(18).…”