Proton translocation coupled to ubiquinol oxidation in Paracoccus denitrificans

Abstract

Lawford, H. G . (1979) Proton translocation coupled to ubiquinol oxidation in Paracoccus denitrificans. Can. J. Biochem. 57,[172][173][174][175][176][177] Measurements were made of the stoichiometry of proton translocation associated with electron flow through the cytochrome-dependent region of the aerobically induced respiratory chain of Paracoccus denitrificans using the pulse oxidant method and rotenone to inhibit NADH dehydrogenase activity. Paracoccus denitrificans (ATCC 13543 ) was grown aerobically in carbon-limited continuous culture ( D = 0.27 h,-I D, dilution rate per hour) with succinate as sole carbon and energy source. Oxidation of exogenous ubiquinol, by starved cells was significantly accelerated by treatment of the cells with lysozyme. Spectrophotometric assay of ubiquinol, oxidase activity in rotenone-poisoned spheroplasts revealed an apparent K, of 25 pM and a Vmnx of 217 nmol/min per milligram protein.Can. J. Biochem. Downloaded from www.nrcresearchpress.com by Queens University on 01/05/15For personal use only.( 37) but by inhibiting the oxidation of substrates (e.g., L-malate) which are produced as a consequence of succinate oxidation and whose oxidation is dependent on Can. J. Biochem. Downloaded from www.nrcresearchpress.com by Queens University on 01/05/15For personal use only.

Cytochrome o (bo) is a proton pump in Paracoccus denitrificans and Escherichia coli

“…Spheroplasts from wild-type Paracoccus cells translocate 2.6-2.9 H÷/e -linked to succinate oxidation by 02 ( fig.lA), confirming the earlier report by Lawford [13]. With ferricyanide as oxidant, accepting electrons at the cytochrome c level, the ratio fell by approx.…”

The efficiency of energy conservation in cytochrome c-deficient mutants of Paracoccus denitrificans

“…In glycerol-limited cells, the +H+: O ratio associated with the oxidation of endogenous substrate has been measured at 8.3 -+ 0.3 (this study) and 8.4 -+ 0.4 [4]. After carbon-limited growth on succinate, ~H÷: O ratios have been measured of 8.5 -+ 0.5 in cells oxidising endogenous substrate [5], 5.9 + 0.1 in cells oxidising endogenous substrate in the presence of the site I-specific inhibitor piericidin A [5], and 5.2 +-0.2 in spheroplasts oxidising ubiquinol, [6]. Van Verseveld and Stouthamer [21] found that carbon-limited cells grown on methanol, in contrast to cells grown on other substrates [8,9], appeared to contain 3 functional sites of energy conservation, and-+H*: O ratios or 7-10.5 were measured in methanol-limited cells oxidising endogenous substrate [10].…”

“…denitrificans is the subject of some controversy. Measurements of molar growth yields in continuous culture, and the stoichiometry of respiration-driven proton translocation (~H+:O), have been variously interpreted as indicating the presence of 3 potential sites of energy conservation, together with an energylinked pyridine nucleotide transhydrogenase [3,4], 3 sites [5][6][7] or only 2 sites [8,9], in heterotropically grown cells. Correspondingly, the ~H÷: site ratio in these cells has been estimated to be 2, as required by the classical chemiosmotic hypothesis [3,4], 3 [5][6][7] or 3-4 [8,9].…”

A two-subunit cytochrome c oxidase (cytochrome aa3) from Paracoccus dentrificans.

“…One intriguing possibility would be that they are necessary for the protonpumping activity of the enzyme and that the bacterial enzymes cannot perform this function, even though quantitative measurements with Paracoccus cells suggest that the H+/e-ratio in this region of the chain is higher than 1 (31).…”