The lipid head group is the key element for substrate recognition by the P4 ATPase ALA2: a phosphatidylserine flippase.

Type IV P-type ATPases (P4 ATPases) are lipid flippases that catalyze phospholipid transport from the exoplasmic to the cytoplasmic leaflet of cellular membranes, but the mechanism by which they recognize and transport phospholipids through the lipid bilayer remains unknown. In the present study, we succeeded in purifying recombinant aminophospholipid ATPase 2 (ALA2), a member of the P4 ATPase subfamily in , in complex with the ALA-interacting subunit 5 (ALIS5). The ATP hydrolytic activity of the ALA2-ALIS5 complex was stimulated in a highly specific manner by phosphatidylserine.

Proton pumping by an inactive structural variant of cytochrome c oxidase.

The aa3-type cytochrome c oxidases (CytcOs) from e.g. Rhodobacter sphaeroides and Paracoccus denitrificans harbor two proton-transfer pathways.

The membrane modulates internal proton transfer in cytochrome c oxidase.

The functionality of membrane proteins is often modulated by the surrounding membrane. Here, we investigated the effect of membrane reconstitution of purified cytochrome c oxidase (CytcO) on the kinetics and thermodynamics of internal electron and proton-transfer reactions during O(2) reduction. Reconstitution of the detergent-solubilized enzyme in small unilamellar soybean phosphatidylcholine vesicles resulted in a lowering of the pK(a) in the pH dependence profile of the proton-uptake rate.

Characterization of a Listeria monocytogenes Ca(2+) pump: a SERCA-type ATPase with only one Ca(2+)-binding site.

We have characterized a putative Ca(2+)-ATPase from the pathogenic bacterium Listeria monocytogenes with the locus tag lmo0841. The purified and detergent-solubilized protein, which we have named Listeria monocytogenes Ca(2+)-ATPase 1 (LMCA1), performs a Ca(2+)-dependent ATP hydrolysis and actively transports Ca(2+) after reconstitution in dioleoylphosphatidyl-choline vesicles. Despite a high sequence similarity to the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1a) and plasma membrane Ca(2+)-ATPase (PMCA), LMCA1 exhibits important biochemical differences such as a low Ca(2+) affinity (K(0.

Charge transfer in the K proton pathway linked to electron transfer to the catalytic site in cytochrome c oxidase.

Cytochrome c oxidase couples electron transfer from cytochrome c to O 2 to proton pumping across the membrane. In the initial part of the reaction of the reduced cytochrome c oxidase with O 2, an electron is transferred from heme a to the catalytic site, parallel to the membrane surface. Even though this electron transfer is not linked to proton uptake from solution, recently Belevich et al.

The inside pH determines rates of electron and proton transfer in vesicle-reconstituted cytochrome c oxidase.

Cytochrome c oxidase is the terminal enzyme in the respiratory chains of mitochondria and many bacteria where it translocates protons across a membrane thereby maintaining an electrochemical proton gradient. Results from earlier studies on detergent-solubilized cytochrome c oxidase have shown that individual reaction steps associated with proton pumping display pH-dependent kinetics. Here, we investigated the effect of pH on the kinetics of these reaction steps with membrane-reconstituted cytochrome c oxidase such that the pH was adjusted to different values on the inside and outside of the membrane.

Inhibition of proton pumping by zinc ions during specific reaction steps in cytochrome c oxidase.

Cytochrome c oxidase (CytcO) is a redox-driven proton pump in the respiratory chain of mitochondria and many aerobic bacteria. The results from several studies have shown that zinc ions interfere with both the uptake and release of protons, presumably by binding near the orifice of the proton entrance and exit pathways. To elucidate the effect of Zn2+ binding on individual electron and proton-transfer reactions, in this study, we have investigated the reaction of the fully reduced R.

The timing of proton migration in membrane-reconstituted cytochrome c oxidase.

In mitochondria and aerobic bacteria energy conservation involves electron transfer through a number of membrane-bound protein complexes to O2. The reduction of O2, accompanied by the uptake of substrate protons to form H2O, is catalyzed by cytochrome c oxidase (CcO). This reaction is coupled to proton translocation (pumping) across the membrane such that each electron transfer to the catalytic site is linked to the uptake of two protons from one side and the release of one proton to the other side of the membrane.

A mechanistic principle for proton pumping by cytochrome c oxidase.

In aerobic organisms, cellular respiration involves electron transfer to oxygen through a series of membrane-bound protein complexes. The process maintains a transmembrane electrochemical proton gradient that is used, for example, in the synthesis of ATP. In mitochondria and many bacteria, the last enzyme complex in the electron transfer chain is cytochrome c oxidase (CytcO), which catalyses the four-electron reduction of O2 to H2O using electrons delivered by a water-soluble donor, cytochrome c.