The two transmembrane helices of CcoP are sufficient for assembly of the cbb3-type heme-copper oxygen reductase from Vibrio cholerae

Abstract

The C-family (cbb3) of heme-copper oxygen reductases are proton-pumping enzymes terminating the aerobic respiratory chains of many bacteria, including a number of human pathogens. The most common form of these enzymes contains one copy each of 4 subunits encoded by the ccoNOQP operon. In the cbb3 from Rhodobacter capsulatus, the enzyme is assembled in a stepwise manner, with an essential role played by an assembly protein CcoH. Importantly, it has been proposed that a transient interaction between the transmembrane domains of CcoP and CcoH is essential for assembly. Here, we test this proposal by showing that a genetically engineered form of cbb3 from Vibrio cholerae (CcoNOQPX) that lacks the hydrophilic domain of CcoP, where the two heme c moieties are present, is fully assembled and stable. Single-turnover kinetics of the reaction between the fully reduced CcoNOQPX and O2 are essentially the same as the wild type enzyme in oxidizing the 4 remaining redox-active sites. The enzyme retains approximately 10% of the steady state oxidase activity using the artificial electron donor TMPD, but has no activity using the physiological electron donor cytochrome c4, since the docking site for this cytochrome is presumably located on the absent domain of CcoP. Residue E49 in the hydrophobic domain of CcoP is the entrance of the KC-channel for proton input, and the E49A mutation in the truncated enzyme further reduces the steady state activity to less than 3%. Hence, the same proton channel is used by both the wild type and truncated enzymes.

Mechanism of proton transfer through the KC proton pathway in the Vibrio cholerae cbb3 terminal oxidase

“…Y227F, Y321F and N293L showed normal UV-visible absorption spectra (Figure S1A) [6]. In the CO-recombination kinetics, wildtype (Wt) V. cholerae cbb 3 shows two kinetic components (described in ref [22], see also Figure S1B); a rapid phase (τ ~60 μs) attributed to CO rebinding to one of the c -type hemes, and a slower phase (τ= 2.2 ms) associated with CO rebinding to the heme b 3 . Both these phases are present in all three variants, with similar time constants as in Wt.…”

“…Reconstitution of the V. cholera cbb 3 and ATP synthase into liposomes was done by mixing lipid vesicles containing ~1% sodium cholate with the V. cholera cbb 3 alone (as in [22]) or together with ATP synthase as in [23]. After incubation for 30 min at room temperature with occasional gentle shaking, the sodium cholate was removed by addition of Bio-beads (BioRad) [22] or gel filtration (on a PD-10 column, GE Healthcare).…”

“…The enzyme samples were prepared as previously described [6, 22]. Briefly, the enzyme at a concentration of ~5 μM was transferred to a modified anaerobic cuvette and the atmosphere was exchanged to N 2 on a vacuum line.…”

“…Flow-flash measurements were performed using a modified stopped-flow apparatus (Applied Photophysics, Surrey, U.K.) essentially as in [24] and data analyzed as described in [6, 22]. The enzyme solution was rapidly mixed in a 1:5 ratio with an O 2- saturated buffer, and the reaction with O 2 was initiated by flash photolysis of the CO-enzyme complex approximately 200 ms after mixing.…”

The Role of the Regulator Fur in Gene Regulation and Virulence of Riemerella anatipestifer Assessed Using an Unmarked Gene Deletion System

“…This inhibition regulates the conversion of available serine to siderophore, enterobactin, which thereby increases iron acquisition (Salvail et al, 2010 ). The cytochrome c oxidase (Cco) family related to oxidation-reduction was also inhibited in response to iron limitation, which is comprised of four subunits, CcoN, CcoO, CcoP, and CcoQ, which act as the terminal enzyme of respiratory chain (Ahn et al, 2015 ; Steininger et al, 2016 ). The Cco family, a member of heme-copper oxidase superfamily, may play a role in iron- restricted conditions.…”