Formation of pH and potential gradients by the reconstituted Azotobacter vinelandii cytochrome bd respiratory protection oxidase

Abstract

To directly characterize the bioenergetic properties of the cytochrome bd terminating branch of the Azotobacter vinelandii electron transport chain, the purified cytochrome bd oxidase was reconstituted into a phospholipid environment consisting of phosphatidylethanolamine and phosphatidylglycerol (3:1). The average diameter of the proteoliposomes after extrusion through a polycarbonate membrane was 94 ؎ 4 nm. Initiation of respiration upon the addition of 20 M ubiquinone-1 to proteoliposomes loaded with the pH-sensitive dye pyranine resulted in an immediate alkalization of the vesicle lumen by an average pH change of 0.11 unit. This pH gradient was readily collapsed upon the addition of nigericin, carbonyl cyanide p-(tri-fluoromethoxy) phenyl-hydrazone, gramicidin, Triton X-100, or 2-heptyl-4-hydroxyquinoline N-oxide (HQNO). Proteoliposomal respiration initiated in the presence of the potentiometric membrane dye rhodamine 123 caused the generation of a transmembrane potential; the potential was collapsed upon the addition of either valinomycin or HQNO. The formation of both pH and potential gradients during turnover demonstrates that the A. vinelandii cytochrome bd oxidase is coupled to energy conservation in vivo.

The cytochrome bd respiratory oxygen reductases

“…The bioenergetic function of cytochrome bd is to conserve energy in the form of Δμ H + [41,45–50], although the H + /e − ratio is 1, half the value of the A-subfamily heme-copper oxygen reductases such as the mitochondrial cytochrome c oxidase or cytochrome bo 3 from E. coli because the bd- type oxygen reductases do not pump protons [45,49,50]. …”

Accommodation of CO in the di-heme active site of cytochrome bd terminal oxidase from Escherichia coli

“…The third heme, low-spin hexacoordinate b 558 , transfers electrons from the natural electron donor quinol towards the di-heme site [22]. Cytochromes bd can generate proton-motive force but without invoking a 'proton pump' mechanism [18,[23][24][25][26][27][28]. They are widely distributed in pathogenic bacteria and responsible for a number of vitally important physiological functions [29][30][31] which include conferring resistance to NO stress [32][33][34][35][36].…”

Time-resolved electrometric and optical studies on cytochrome bd suggest a mechanism of electron-proton coupling in the di-heme active site

“…Unlike the heme-copper oxidases (4,5), cytochrome bd does not pump protons (6). However, the oxidation of ubiquinol by O 2 catalyzed by cytochrome bd is linked to the generation of transmembrane electric potential difference (⌬⌿) (7)(8)(9)(10)(11) by virtue of the fact that the protons resulting from the oxidation of ubiquinol are released into the bacterial periplasm, whereas the protons used to convert O 2 to H 2 O are taken from the bacterial cytoplasm. Cytochrome bd is a heterodimeric enzyme containing three hemes: b 558 , b 595 , and d (12)(13)(14) but no copper.…”