Proton pumping by cytochrome oxidase as studied by time-resolved stopped-flow spectrophotometry.

Abstract

The H+/e-stoichiometry for the proton pump of cytochrome c oxidase reportedly varies between 0 and 1, depending on experimental conditions. In this paper, we report the results obtained by a combination oftransient optical spectroscopy with a time resolution of 10 ms and a singular value decomposition analysis to follow the kinetics, separate the observed spectral components, and quantitate the stoichiometry of the pump. By using cytochrome oxidase reconstituted into small unilamellar vesicles, we show that the time courses of ferrocytochrome c oxidation and phenol red acidification or alk tion fit a simple kinetic scheme. The fitting procedure leads to unbiased and objective determination of the H+/eratio under various experimental conditions. The protonpumping stoichiometry was found to be 1.01 ± 0.10, independent ofthe number ofturnovers, proton back-leak rate, or type of experiment (oxidant or reductant pulse). (6) have been carried out by measuring external acidification by potentiometry; with this method, it is very difficult to follow the time course of proton pumping, especially during the initial turnovers. On the other hand, overlap of the absorption bands of the cytochromes and the pH indicator phenol red (pKa = 7.7) makes it very difficult to objectively analyze the time course of electron transfer and H+ translocation; the outcome relies on calibrations to set proper isosbestic points for a point-bypoint correction of the time-dependent spectral overlaps. Therefore, application of stopped-flow spectrophotometry has been very limited, and the relatively slow-responding potentiometric method has been almost universally employed.In this study, we have employed transient spectroscopy and singular value decomposition (SVD) analysis to follow the time course of the various spectral components and to assess quantitatively the stoichiometry of the proton pump in COVs, under selected conditions. A simple kinetic scheme fits the data at all wavelengths (500-650 nm) and allows us to prove (i) that the H+/e-stoichiometry (1.01 ± 0.10) is independent of the total number of turnovers (from 2 to 10 turnovers) and of the rate of proton back-leak [increased by various concentrations of carbonylcyanide m-chlorophenylhydrazone (CCCP)] and (ii) that experiments initiated either with a pulse of reductant (cytochrome c2+) or with a pulse of oxidant (oxygen) yield the same stoichiometry, proving that Abbreviations: COV, cytochrome c oxidase vesicle; SVD, singular value decomposition; CCCP, carbonylcyanide m-chlorophenylhydrazone. §To whom reprint requests should be addressed at:

Controlled uncoupling and recoupling of proton pumping in cytochrome c oxidase

“…Both the P 2 ͞P 3 3 F 3 and the F 3 3 O 4 transitions are linked to pumping of one proton in each transition (21)(22)(23)(24). In addition, the reduction of the oxidized CcO is presumably also associated with pumping (23,25) maintaining an average proton-pumping stoichiometry of one pumped proton per electron transferred to the catalytic site (26).…”

Kinetic coupling between electron and proton transfer in cytochrome c oxidase: simultaneous measurements of conductance and absorbance changes.

“…It is a membrane-bound protein complex that couples an electron current across the membrane to a proton current in the opposite direction (1) with a H+/e-stoichiometry of one (2). Highresolution x-ray structures of both the bacterial enzyme from Paracoccus denitnificans (3) and the bovine-heart enzyme (4) have recently been determined.…”

Chemiosmotic misunderstandings

“…No sources for the values used (whether proton pumping cost or nunber of protons trasnfered from the matrix) were provided in that publication. A quick estimate (based on a protonmotive force of 200 mV [31]) yields instead a total cost of 36.9 kcal/mol for the extrusion of four protons per electron pair of complex I [32], and two protons per electron pair by each of complex III [33] and complex IV [34], which can be easily provided by the transport of two electrons from NADH to O 2 (1.13 V, i.e. 52 kcal/mol).…”