Controlled uncoupling and recoupling of proton pumping in cytochrome
            <i>c</i>
            oxidase

Brändén, Gisela; Pawate, Ashtamurthy S.; Gennis, Robert B.; Brzezinski, Peter

doi:10.1073/pnas.0507734103

Cited by 65 publications

(65 citation statements)

References 52 publications

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“…As described above, results from earlier studies have shown that introduction of negative charges near the D pathway orifice in the D132N/A mutant oxidases could restore proton pumping and accelerate proton uptake (18,19,39). These data were interpreted to suggest that slowed proton uptake in the D132N single mutant is caused by the removal of negative charge (40).…”

Section: Discussionmentioning

confidence: 65%

“…Rapid proton uptake and pumping could be partly restored by introduction of an Asp at the site of Asn-139, located ∼7 Å from Asp-132 (N139D/ D132N double mutation) (18,19). Collectively, these results were interpreted to suggest that rapid proton uptake to the D pathway (and proton pumping) cannot be maintained if Asp-132 is removed without introduction of a compensatory negative charge in the vicinity.…”

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confidence: 79%

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

Johansson

Högbom

Carlsson

et al. 2013

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

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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.

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Section: Discussionmentioning

confidence: 65%

mentioning

confidence: 79%

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

Johansson

Högbom

Carlsson

et al. 2013

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

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“…34 The release of cytochrome c from mitochondria to the cytoplasm can trigger apoptosis in osteoblasts. 35 Oxidative stress increased the cellular levels of cytochrome c protein. Such an increase put osteoblasts at a high risk of cell apoptosis.…”

Section: Discussionmentioning

confidence: 99%

Nitric oxide protects osteoblasts from oxidative stress‐induced apoptotic insults via a mitochondria‐dependent mechanism

Chang

Liao

Lin

et al. 2006

Journal Orthopaedic Research

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Nitric oxide (NO) contributes to the regulation of osteoblast activities. In this study, we evaluated the protective effects of NO pretreatment on oxidative stress-induced osteoblast apoptosis and its possible mechanism using neonatal rat calvarial osteoblasts as the experimental model. Exposure of osteoblasts to sodium nitroprusside (SNP) at a low concentration of 0.3 mM significantly increased cellular NO levels without affecting cell viability. However, when the concentration reached a high concentration of 2 mM, SNP increased the levels of intracellular reactive oxygen species and induced osteoblast injuries. Thus, administration of 0.3 and 2 mM SNP in osteoblasts were respectively used as sources of NO and oxidative stress. Pretreatment with NO for 24 h significantly ameliorated the oxidative stress-caused morphological alterations and decreases in alkaline phosphatase activity, and reduced cell death. Oxidative stress induced osteoblast death via an apoptotic mechanism, but NO pretreatment protected osteoblasts against the toxic effects. The mitochondrial membrane potential was significantly reduced following exposure to the oxidative stress. However, pretreatment with NO significantly lowered the suppressive effects. Oxidative stress increased cellular Bax protein production and cytochrome c release from mitochondria. Pretreatment with NO significantly decreased oxidative stress-caused augmentation of Bax and cytochrome c protein levels. In parallel with cytochrome c release, oxidative stress induced caspase-3 activation and DNA fragmentation. Pretreatment with NO significantly reduced the oxidative stress-enhanced caspase-3 activation and DNA damage. Results of this study show that NO pretreatment can protect osteoblasts from oxidative stress-induced apoptotic insults. The protective action involves a mitochondria-dependent mechanism. ß

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“…The significance of the increase of the pK a of E286, measured by the pH-dependence of the P R →F transition, was given further support by the subsequent study of the double mutant N139D/D132N, in which it was shown that replacing the aspartic acid at position D132 by an asparagine restores the proton pumping abolished by the N139D mutation (11). Of particular interest is that the pH-dependence of the P R →F transition is also restored to a value near that of the wild type oxidase.…”

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confidence: 96%

Replacing Asn207 by Aspartate at the Neck of the D Channel in the aa₃-Type Cytochrome c Oxidase from Rhodobacter sphaeroides Results in Decoupling the Proton Pump

et al. 2006

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Cytochrome oxidase catalyzes the reduction of O 2 to water and conserves the considerable free energy available from this reaction in the form of a proton motive force. For each electron, one proton is electrogenically pumped across the membrane. Of particular interest is the mechanism by which the proton pump operates. Previous studies of the oxidase from Rhodobacter sphaeroides have shown that all the pumped protons enter the enzyme through the D channel, and that a point mutant, N139D, in the D channel completely eliminates proton pumping without reducing the oxidase activity. N139 is one of three asparagines near the entrance of the D channel where there is a narrowing or neck, through which a single file of water molecules pass. In the current work, it is shown that replacement of a second asparagine in this region by an asparate, N207D, also decouples the proton pump without altering the oxidase activity of the enzyme. Previous studies demonstrated that the N139D mutant results in an increase in the apparent pK a of E286, a functionally critical residue which is located 20 Å away from N139 at the opposite end of the D channel. In the current work, it is shown that the N207 mutation also increases the apparent pK a of E286. This finding re-enforces the proposal that the elimination of proton pumping is the result of an increase of the apparent proton affinity of E286 which, in turn, prevents the timely proton transfer to a proton accepter group within the exit channel of the proton pump.During each turnover of cytochrome c oxidase, eight protons are taken up from the N-side of the membrane; four protons are used for chemistry (water formation) and four protons are pumped across the membrane(1-6). Recently, a mutation in the D-channel of the aa 3 -type oxidase from Rhodobacter sphaeroides, N139D (see Figure 1), was shown to completely eliminate proton pumping, but without reducing the oxidase activity of the enzyme (7-9). Indeed, the mutant oxidase has a turnover number that is twice that of the wild type enzyme. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2008 September 12. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptA similar mutation has also been reported at the equivalent position (N131D) in the oxidase from Paracoccus denitrificans (10). The N139D mutant of theR. sphaeroides oxidase has been further characterized and shown to form the same transient intermediates as observed with the wild type oxidase (8). In this reaction, O 2 binds to the reduced heme a 3 -Cu B active site and the O-O bond is split yielding hydroxide associated with Cu B 2+ and an oxoferryl form (Fe 4+ =O 2− ) of heme a 3 . This reaction involves the transfer of one electron from nearby heme a, which leaves the active site with excess negative charge. This intermediate is called the P R state of the oxidase, and it is shortlived (τ< 100 μs). The P R state is rapidly converted to the F state upon the transfer of a proton from E286 to the active site. Hence...

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Controlled uncoupling and recoupling of proton pumping in cytochrome c oxidase

Cited by 65 publications

References 52 publications

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

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

Nitric oxide protects osteoblasts from oxidative stress‐induced apoptotic insults via a mitochondria‐dependent mechanism

Replacing Asn207 by Aspartate at the Neck of the D Channel in the aa₃-Type Cytochrome c Oxidase from Rhodobacter sphaeroides Results in Decoupling the Proton Pump

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Controlled uncoupling and recoupling of proton pumping in cytochrome c oxidase

Cited by 65 publications

References 52 publications

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

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

Nitric oxide protects osteoblasts from oxidative stress‐induced apoptotic insults via a mitochondria‐dependent mechanism

Replacing Asn207 by Aspartate at the Neck of the D Channel in the aa3-Type Cytochrome c Oxidase from Rhodobacter sphaeroides Results in Decoupling the Proton Pump

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Replacing Asn207 by Aspartate at the Neck of the D Channel in the aa₃-Type Cytochrome c Oxidase from Rhodobacter sphaeroides Results in Decoupling the Proton Pump