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

Abstract

Reduction of trimethylamine N-oxide (E 0(TMAO/TMA) ‫؍‬ ؉130 mV) in Escherichia coli is carried out by the Tor system, an electron transfer chain encoded by the torCAD operon and made up of the periplasmic terminal reductase TorA and the membrane-anchored pentahemic c-type cytochrome TorC. Although the role of TorA in the reduction of trimethylamine N-oxide (TMAO) has been clearly established, no direct evidence for TorC involvement has been presented. TorC belongs to the NirT/NapC c-type cytochrome family based on homologies of its N-terminal tetrahemic domain (TorC N ) to the cytochromes of this family, but TorC contains a C-terminal extension (TorC C ) with an additional hemebinding site. In this study, we show that both domains are required for the anaerobic bacterial growth with TMAO. The intact TorC protein and its two domains, TorC N and TorC C , were produced independently and purified for a biochemical characterization. The reduced form of TorC exhibited visible absorption maxima at 552, 523, and 417 nm. Mediated redox potentiometry of the heme centers of the purified components identified two negative midpoint potentials (؊177 and ؊98 mV) localized in the tetrahemic TorC N and one positive midpoint potential (؉120 mV) in the monohemic TorC C . In agreement with these values, the in vitro reconstitution of electron transfer between TorC, TorC N , or TorC C and TorA showed that only TorC and TorC C were capable of electron transfer to TorA. Surprisingly, interaction studies revealed that only TorC and TorC N strongly bind TorA. Therefore, TorC C directly transfers electrons to TorA, whereas TorC N , which probably receives electrons from the menaquinone pool, is involved in both the electron transfer to TorC C and the binding to TorA.

An unsuspected autoregulatory pathway involving apocytochrome TorC and sensor TorS in Escherichia coli

“…The mature TorC C domain is the direct electron donor to TorA, and the good positioning of TorC C on TorA seems to result from the strong interaction between TorA and the TorC N domain (Fig. 7) (8). If apoTorC could still bind to TorA, then apoTorC C could not be available to interact with TorS.…”

“…The N-terminal domain (TorC N ) contains a membrane anchor and four hemebinding sites, whereas the C-terminal domain (TorC C ) possesses only one heme-binding motif (8). As each TorC domain contains heme-binding sites, negative regulation, which depends on the absence of heme insertion, might require only one of the two domains of TorC.…”

“…Once matured, TorC tightly binds periplasmic TorA and shuttles electrons from the menaquinones to TorA (8). To better understand the dual role of TorC according to its maturation state, we investigated whether the immature form of TorC could still bind the TorA enzyme.…”

“…We recently showed that TorC is an essential component of the TMAO respiratory chain and that it shuttles electrons from the membranous menaquinones to TorA. Surprisingly, the TorC N domain not only transfers electrons to the TorC C domain but also binds to TorA; in contrast, TorC C contains the electron donor site to TorA but is not involved in TorA binding (8). Like other c-type cytochromes, TorC is synthesized first as an immature cytochrome (apocytochrome) that must cross the inner membrane via the Sec pathway before maturation occurs in the periplasm (10).…”

“…TorC is anchored to the inner membrane by a hydrophobic N-terminal extremity but its main body, which comprises two domains of equivalent size (about 20 kDa), is in the periplasm (8). The N-terminal domain of TorC (TorC N ) contains four heme-binding motifs CXXCH and is highly homologous to the members of the large NirT͞NapC family of c-type cytochromes (9).…”