Partial Steps of Charge Translocation in the Nonpumping N139L Mutant of <i>Rhodobacter sphaeroides</i> Cytochrome <i>c</i> Oxidase with a Blocked D-Channel

Siletsky, Sergey A.; Zhu, Jiapeng; Gennis, Robert B.; Konstantinov, Alexander A.

doi:10.1021/bi901719e

Cited by 31 publications

(35 citation statements)

References 92 publications

(312 reference statements)

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“…As a result, the subsequent PT from E286 to the BNC that converts the F state to the O state will also be slowed down. This is consistent with the experimental data that the F → O transition is significantly slowed down in this mutant (16). A closer inspection of the 2D-PMF (Fig.…”

Section: N139l Mutant Inhibits Reprotonation Of E286 Through the D-chsupporting

confidence: 92%

“…This would eliminate proton pumping. In the N139L mutant, the proton pumping is essentially eliminated (16), which supports our prediction. Moreover, the slowdown of E286 reprotonation will leave the E286 in its deprotonated form for a longer time in the F state (16).…”

Section: N139l Mutant Inhibits Reprotonation Of E286 Through the D-chsupporting

confidence: 89%

“…In the N139L mutant, the proton pumping is essentially eliminated (16), which supports our prediction. Moreover, the slowdown of E286 reprotonation will leave the E286 in its deprotonated form for a longer time in the F state (16). As a result, the subsequent PT from E286 to the BNC that converts the F state to the O state will also be slowed down.…”

Section: N139l Mutant Inhibits Reprotonation Of E286 Through the D-chsupporting

confidence: 89%

“…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%

“…S1A). It is the mutation of N139 (into various amino acid alternatives) that decouples proton pumping from chemical reaction most often (9)(10)(11)(14)(15)(16)(17)(18). However, how the mutation of N139 influences the key pumping and chemical reaction PT steps, which occur above E286 more than 25 Å away ( Fig.…”

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confidence: 99%

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

Liang

Swanson

Wikström

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Section: N139l Mutant Inhibits Reprotonation Of E286 Through the D-chsupporting

confidence: 92%

Section: N139l Mutant Inhibits Reprotonation Of E286 Through the D-chsupporting

confidence: 89%

Section: N139l Mutant Inhibits Reprotonation Of E286 Through the D-chsupporting

confidence: 89%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

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

Liang

Swanson

Wikström

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Application of direct electrometry in studies of microbial rhodopsins reconstituted in proteoliposomes

et al. 2022

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Time-resolved single-turnover of caa3 oxidase from Thermus thermophilus. Fifth electron of the fully reduced enzyme converts OH into EH state

Siletsky

Belevich

et al. 2011

Biochimica et Biophysica Acta (BBA) - Bioenergetics

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The oxidative part of the catalytic cycle of the caa(3)-type cytochrome c oxidase from Thermus thermophilus was followed by time-resolved optical spectroscopy. Rate constants, chemical nature and the spectral properties of the catalytic cycle intermediates (Compounds A, P, F) reproduce generally the features typical for the aa(3)-type oxidases with some distinctive peculiarities caused by the presence of an additional 5-th redox-center-a heme center of the covalently bound cytochrome c. Compound A was formed with significantly smaller yield compared to aa(3) oxidases in general and to ba(3) oxidase from the same organism. Two electrons, equilibrated between three input redox-centers: heme a, Cu(A) and heme c are transferred in a single transition to the binuclear center during reduction of the compound F, converting the binuclear center through the highly reactive O(H) state into the final product of the reaction-E(H) (one-electron reduced) state of the catalytic site. In contrast to previous works on the caa(3)-type enzymes, we concluded that the finally produced E(H) state of caa(3) oxidase is characterized by the localization of the fifth electron in the binuclear center, similar to the O(H)→E(H) transition of the aa(3)-type oxidases. So, the fully-reduced caa(3) oxidase is competent in rapid electron transfer from the input redox-centers into the catalytic heme-copper site.

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Partial Steps of Charge Translocation in the Nonpumping N139L Mutant of Rhodobacter sphaeroides Cytochrome c Oxidase with a Blocked D-Channel

Cited by 31 publications

References 92 publications

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

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

Application of direct electrometry in studies of microbial rhodopsins reconstituted in proteoliposomes

Time-resolved single-turnover of caa3 oxidase from Thermus thermophilus. Fifth electron of the fully reduced enzyme converts OH into EH state

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