Single Enzyme Experiments Reveal a Long-Lifetime Proton Leak State in a Heme-Copper Oxidase

Abstract

Heme-copper oxidases (HCOs) are key enzymes in prokaryotes and eukaryotes for energy production during aerobic respiration. They catalyze the reduction of the terminal electron acceptor, oxygen, and utilize the Gibbs free energy to transport protons across a membrane to generate a proton (ΔpH) and electrochemical gradient termed proton motive force (PMF), which provides the driving force for the adenosine triphosphate (ATP) synthesis. Excessive PMF is known to limit the turnover of HCOs, but the molecular mechanism of this regulatory feedback remains relatively unexplored. Here we present a single-enzyme study that reveals that cytochrome bo3 from Escherichia coli, an HCO closely homologous to Complex IV in human mitochondria, can enter a rare, long-lifetime leak state during which proton flow is reversed. The probability of entering the leak state is increased at higher ΔpH. By rapidly dissipating the PMF, we propose that this leak state may enable cytochrome bo3, and possibly other HCOs, to maintain a suitable ΔpH under extreme redox conditions.

Single Proteoliposomes with E. coli Quinol Oxidase: Proton Pumping without Transmembrane Leaks

“…bo 3 was studied at single‐molecule level. The results from these studies suggested that an oxidase molecule could enter a “long‐lifetime leak state” during turnover . This is a surprising observation because a proton leak across any of the cyt.…”

“…bo 3 per liposome. While in the earlier study electrons were supplied to the cyt. bo 3 via a gold electrode, in the present study the electrons were supplied from ubiquinol (UQ 1 ) and the reducing agent dithiothreitol (DTT).…”

Direct observation of proton pumping by a eukaryotic P-type ATPase

“…Patch clamp recording has made it possible to observe the functional dynamics of single ion channels revealing discrete on and off states, subconductance states, and other mechanistically important features that macroscopic experiments cannot probe (2). However, despite extensive structural and biochemical efforts (3), we currently lack a similar depth of understanding of transporters because they in general do not produce electrically detectable single-molecule transport signals (4–8). Here we monitored at the single-molecule level the functional dynamics of a eukaryotic primary active transporter, Arabidopsis thaliana H + -ATPase isoform 2 (AHA2, referred to as the proton pump), which is responsible for energizing the plasma membrane of plants and fungi (Fig.…”

Determining the Attenuation Factor in Molecular Wires Featuring Covalent and Noncovalent Tectons

“…[9] Inspired by this finding,w e present herein as et of noncovalently associated C 60 -pphenylene oligomers,i nw hich the p-conjugation length of the oligomers in the bridges has been systematically altered. [11] From as ynthetic perspective,a symmetric p-phenylene oligomers with aldehyde and carboxylic termini are the key intermediates en route to pyrrolidino[3,4:1,2][60]fullerenes 1a-d.T hese oligomers were synthesized by Suzuki crosscoupling reactions between bromoaryl and organoborane compounds in the presence of ap alladium(II) catalyst and afluorine salt as abase and additive,which is crucial for the success of the cross-coupling reaction. On the basis of this molecular design, we determined the effects of distance and the rate at which electron-transfer processes occur,a sw ell as the molecularwire behavior of systems based on p conjugation and hydrogen bonds.T his bioinspired approach combining electron transfer through supramolecular and covalent connectivity resembles charge transport in av ariety of natural processes, such as photosynthesis [10] and cellular respiration.…”

“…On the basis of this molecular design, we determined the effects of distance and the rate at which electron-transfer processes occur,a sw ell as the molecularwire behavior of systems based on p conjugation and hydrogen bonds.T his bioinspired approach combining electron transfer through supramolecular and covalent connectivity resembles charge transport in av ariety of natural processes, such as photosynthesis [10] and cellular respiration. [11] From as ynthetic perspective,a symmetric p-phenylene oligomers with aldehyde and carboxylic termini are the key intermediates en route to pyrrolidino[3,4:1,2][60]fullerenes 1a-d.T hese oligomers were synthesized by Suzuki crosscoupling reactions between bromoaryl and organoborane compounds in the presence of ap alladium(II) catalyst and afluorine salt as abase and additive,which is crucial for the success of the cross-coupling reaction. [12] Subsequent 1,3dipolar cycloaddition reactions were used to link the pphenylene oligomers to C 60 through apyrrolidine ring to yield 1a-d in high yields (see the Supporting Information for synthetic details).…”