Variable proton-pumping stoichiometry in structural variants of cytochrome c oxidase

Self Cite

Proton transfer across biological membranes underpins central processes in biological systems, such as energy conservation and transport of ions and molecules. In the membrane proteins involved in these processes, proton transfer takes place through specific pathways connecting the two sides of the membrane via control elements within the protein. It is commonly believed that acidic residues are required near the orifice of such proton pathways to facilitate proton uptake. In cytochrome c oxidase, one such pathway starts near a conserved Asp-132 residue. Results from earlier studies have shown that replacement of Asp-132 by, e.g., Asn, slows proton uptake by a factor of ∼5,000. Here, we show that proton uptake at full speed (∼10 4 s −1 ) can be restored in the Asp-132–Asn oxidase upon introduction of a second structural modification further inside the pathway (Asn-139–Thr) without compensating for the loss of the negative charge. This proton-uptake rate was insensitive to Zn 2+ addition, which in the wild-type cytochrome c oxidase slows the reaction, indicating that Asp-132 is required for Zn 2+ binding. Furthermore, in the absence of Asp-132 and with Thr at position 139, at high pH (>9), proton uptake was significantly accelerated. Thus, the data indicate that Asp-132 is not strictly required for maintaining rapid proton uptake. Furthermore, despite the rapid proton uptake in the Asn-139–Thr/Asp-132–Asn mutant cytochrome c oxidase, proton pumping was impaired, which indicates that the segment around these residues is functionally linked to pumping.

Section: Discussionmentioning

confidence: 99%

Role of aspartate 132 at the orifice of a proton pathway in cytochromecoxidase

Johansson

Högbom

Carlsson

et al. 2013

Self Cite

“…In these variants the lower stoichiometry could be explained in terms of a competition of proton transfers from a protonatable site (Glu286) within the D proton pathway to a pump site and the catalytic site, respectively (20). In this case the proton-pumping stoichiometry can adopt any value 0-1 depending on the relative rates of internal proton transfers to these two sites.…”

mentioning

confidence: 99%

“…When addressing this problem it is essential to investigate the individual electron-and proton-transfer reactions in the CytcOs (for review, see, e.g., ref. 20). In a representative experiment, all four electrons needed for reduction of O 2 to H 2 O are preloaded into the four redoxactive sites (Cu A , heme a∕b, heme a 3 , and Cu B ) of these enzymes in the absence of O 2 but in the presence of carbon monoxide, which binds to the reduced heme a 3 .…”

mentioning

confidence: 99%

Kinetic design of the respiratory oxidases

Ballmoos

Gennis²,

Ädelroth

et al. 2011

Self Cite

Energy conservation in all kingdoms of life involves electron transfer, through a number of membrane-bound proteins, associated with proton transfer across the membrane. In aerobic organisms, the last component of this electron-transfer chain is a respiratory heme-copper oxidase that catalyzes reduction of O 2 to H 2 O, linking this process to transmembrane proton pumping. So far, the molecular mechanism of proton pumping is not known for any system that is driven by electron transfer. Here, we show that this problem can be addressed and elucidated in a unique cytochrome c oxidase (cytochrome ba 3 ) from a thermophilic bacterium, Thermus thermophilus. The results show that in this oxidase the electron-and proton-transfer reactions are orchestrated in time such that previously unresolved proton-transfer reactions could be directly observed. On the basis of these data we propose that loading of the proton pump occurs upon electron transfer, but before substrate proton transfer, to the catalytic site. Furthermore, the results suggest that the pump site alternates between a protonated and deprotonated state for every second electron transferred to the catalytic site, which would explain the noninteger pumping stoichiometry (0.5 H þ ∕e − ) of the ba 3 oxidase. Our studies of this variant of Nature's palette of mechanistic solutions to a basic problem offer a route toward understanding energy conservation in biological systems.rapid kinetics | membrane protein | electrochemical gradient

“…1), is unknown. It is speculated that replacement with a charged residue, such as N139D, induces decoupling by shifting the pK a of the E286 via long-range electrostatic interactions (e.g., with the negatively charged D139 residue) (20,21). However, proton pumping can also be decoupled by the replacement of N139 with charge-neutral residues (e.g., N139T, N139C, and N139L), suggesting a more nuanced decoupling mechanism for these mutants.…”

mentioning

confidence: 99%

“…The decoupling mutants maintain the O 2 reduction reaction [often at the same rate as the wild-type (WT) CcO], but fail to couple the chemical reaction to proton pumping (9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). Mutagenesis studies have further revealed several residues in the D-channel that are essential for efficient proton pumping.…”

mentioning

confidence: 99%

Understanding the essential proton-pumping kinetic gates and decoupling mutations in cytochrome c oxidase

Liang

Swanson

Wikström

et al. 2017

Cytochrome c oxidase (CcO) catalyzes the reduction of oxygen to water and uses the released free energy to pump protons against the transmembrane proton gradient. To better understand the proton-pumping mechanism of the wild-type (WT) CcO, much attention has been given to the mutation of amino acid residues along the proton translocating D-channel that impair, and sometimes decouple, proton pumping from the chemical catalysis.Although their influence has been clearly demonstrated experimentally, the underlying molecular mechanisms of these mutants remain unknown. In this work, we report multiscale reactive molecular dynamics simulations that characterize the free-energy profiles of explicit proton transport through several important D-channel mutants. Our results elucidate the mechanisms by which proton pumping is impaired, thus revealing key kinetic gating features in CcO. In the N139T and N139C mutants, proton back leakage through the D-channel is kinetically favored over proton pumping due to the loss of a kinetic gate in the N139 region. In the N139L mutant, the bulky L139 side chain inhibits timely reprotonation of E286 through the D-channel, which impairs both proton pumping and the chemical reaction. In the S200V/S201V double mutant, the proton affinity of E286 is increased, which slows down both proton pumping and the chemical catalysis. This work thus not only provides insight into the decoupling mechanisms of CcO mutants, but also explains how kinetic gating in the D-channel is imperative to achieving high proton-pumping efficiency in the WT CcO.proton pump | decoupling mutants | cytochrome c oxidase | proton transport | multiscale C ytochrome c oxidase (CcO) is the terminal enzyme of the respiratory electron transfer chain in the inner membrane of mitochondria and the plasma membrane of bacteria. It catalyzes the oxidation of cytochrome c molecules and reduction of O 2 to H 2 O. For the aa 3 -type CcO, found in mitochondria and many bacteria, the free energy available from the chemical reaction is used to pump one proton across the membrane per electron transferred to O 2 , generating the transmembrane electrochemical proton gradient necessary for ATP synthesis (1-3). Thus, four "substrate" protons are taken up from the mitochondrial matrix or bacterial cytoplasmic side (negatively charged; N-side) of the membrane and consumed in the reduction of O 2 , whereas another four "pumped" protons are transported to the intermembrane (or extracellular in bacteria) space (positively charged; P-side) of the membrane (Fig. 1). All four pumped protons and at least two of the substrate protons are taken up from solution on the N-side of the membrane by the D-channel, which begins with amino acid residue D132 and ends at residue E286 two-thirds of the way into the protein (4, 5). The remaining substrate protons are taken up by the K-channel (not shown in Fig. 1, but extensively discussed in ref. 6). Next, the protons are either transferred to the binuclear center (BNC) to react with O 2 or to a pump loading site (P...