Abstract: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 memb… Show more
“…During each of these transitions, one proton is released (pumped) and two are taken up (29), resulting in the observed net of 1 proton per transition. The N207D mutant also takes up one proton coincident with the P R →F transition and another proton during the F →O transition.…”
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...
“…During each of these transitions, one proton is released (pumped) and two are taken up (29), resulting in the observed net of 1 proton per transition. The N207D mutant also takes up one proton coincident with the P R →F transition and another proton during the F →O transition.…”
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...
“…After reprotonation of Glu down Ϫ , the final step of the mechanism releases the proton in the PLS to the P-side of the membrane driven by uptake of the chemical proton (Fig. 1), usually on the milliseconds time scale (8,48). This slow reaction creates a situation where prior steps (Fig.…”
Aerobic life is based on a molecular machinery that utilizes oxygen as a terminal electron sink. The membrane-bound cytochrome c oxidase (CcO) catalyzes the reduction of oxygen to water in mitochondria and many bacteria. The energy released in this reaction is conserved by pumping protons across the mitochondrial or bacterial membrane, creating an electrochemical proton gradient that drives production of ATP. A crucial question is how the protons pumped by CcO are prevented from flowing backwards during the process. Here, we show by molecular dynamics simulations that the conserved glutamic acid 242 near the active site of CcO undergoes a protonation state-dependent conformational change, which provides a valve in the pumping mechanism. The valve ensures that at any point in time, the proton pathway across the membrane is effectively discontinuous, thereby preventing thermodynamically favorable proton back-leakage while maintaining an overall high efficiency of proton translocation. Suppression of proton leakage is particularly important in mitochondria under physiological conditions, where production of ATP takes place in the presence of a high electrochemical proton gradient.cell respiration ͉ gating mechanism ͉ proton leak ͉ proton translocation
“…When the flow-flash reaction is performed in H 2 O buffer, the rates of proton uptake and release during the P R →F transition are essentially the same, and they appear coincident in time (Salomonsson et al 2005). This is observed in phospholipid vesicles.…”
Section: Separating Chemical Protons From Pumped Protons By Isotope Ementioning
confidence: 99%
“…This is observed in phospholipid vesicles. When D 2 O solvent is used in place of H 2 O, the rate of proton release is slowed down about sevenfold, whereas the rate of proton uptake is slower by only a factor of about 1.5 (Salomonsson et al 2005). This allows one to clearly observe that the uptake of both the pumped and chemical protons during the P R →F transition can occur prior to proton release.…”
Section: Separating Chemical Protons From Pumped Protons By Isotope Ementioning
Cytochrome c oxidase generates a proton motive force by two separate mechanisms. The first mechanism is similar to that postulated by Peter Mitchell, and is based on electrons and protons used to generate water coming from opposite sides of the membrane. The second mechanism was not initially anticipated, but is now firmly established as a proton pump. A brief review of the current state of our understanding of the proton pump of cytochrome oxidase is presented. We have come a long way since the initial observation of the pump by Mårten Wikström in 1977, but a number of essential questions remain to be answered.
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