Proton transfer during the reaction between fully reduced cytochrome c oxidase and dioxygen: pH and deuterium isotope effects

Cytochrome c oxidase (CcO) is the terminal enzyme of the respiratory chain and couples energetically the reduction of oxygen to water to proton pumping across the membrane. The results from previous studies showed that proton pumping can be uncoupled from the O 2-reduction reaction by replacement of one single residue, Asn-139 by Asp (N139D), located Ϸ30 Å from the catalytic site, in the D-proton pathway. The uncoupling was correlated with an increase in the pK a of an internal proton donor, Glu-286, from Ϸ9.4 to >11. Here, we show that replacement of the acidic residue, Asp-132 by Asn in the N139D CcO (D132N͞N139D double-mutant CcO) results in restoration of the Glu-286 pK a to the original value and recoupling of the proton pump during steady-state turnover. Furthermore, a kinetic investigation of the specific reaction steps in the D132N͞N139D double-mutant CcO showed that proton pumping is sustained even if proton uptake from solution, through the D-pathway, is slowed. However, during single-turnover oxidation of the fully reduced CcO the P 3 F transition, which does not involve electron transfer to the catalytic site, was not coupled to proton pumping. The results provide insights into the mechanism of proton pumping by CcO and the structural elements involved in this process.cytochrome oxidase ͉ electron transfer ͉ gating ͉ proton transfer

Section: Discussionsupporting

confidence: 71%

Controlled uncoupling and recoupling of proton pumping in cytochrome c oxidase

Brändén

Pawate

Gennis

et al. 2006

“…In the presence of 10 mM K 4 further supporting the formation of high-valent Fe IV = O species on the electrode during O 2 reduction. Note that Fe III -OOH species, also produced on the electrode, is proposed to oxidize C-H bonds (46 despite having a much larger driving force (51). Based on the intermediates identified, a mechanistic proposal of O 2 reduction by these Fe porphyrins is shown in Fig.…”

Section: Resultsmentioning

confidence: 97%

Direct observation of intermediates formed during steady-state electrocatalytic O ₂ reduction by iron porphyrins

Sengupta

Chatterjee

Samanta

et al. 2013

158

Heme/porphyrin-based electrocatalysts (both synthetic and natural) have been known to catalyze electrochemical O 2 , H + , and CO 2 reduction for more than five decades. So far, no direct spectroscopic investigations of intermediates formed on the electrodes during these processes have been reported; and this has limited detailed understanding of the mechanism of these catalysts, which is key to their development. Rotating disk electrochemistry coupled to resonance Raman spectroscopy is reported for iron porphyrin electrocatalysts that reduce O 2 in buffered aqueous solutions. Unlike conventional single-turnover intermediate trapping experiments, these experiments probe the system while it is under steady state. A combination of oxidation and spin-state marker bands and metal ligand vibrations (identified using isotopically enriched substrates) allow in situ identification of O 2 -derived intermediates formed on the electrode surface. This approach, combining dynamic electrochemistry with resonance Raman spectroscopy, may be routinely used to investigate a plethora of metalloporphyrin complexes and heme enzymes used as electrocatalysts for small-molecule activation.lectrocatalysis has taken center stage in contemporary research because of its indisputable relevance in the thriving area of renewable energy. Electrocatalytic O 2 reduction, O 2 evolution, H 2 evolution, and H 2 oxidation thus are areas of great interest and have attracted focused effort across several disciplines of science (1-11). Both natural systems [metalloenzymes such as cytochrome c oxidase (CcO), hydrogenase, and glucose oxidase] and biomimetic systems (i.e., synthetic/biochemical mimics of the natural enzyme) are known to function as electrocatalysts (12-15). In particular, it has been known for five decades that heme/porphyrin-based electrocatalysts (both natural and synthetic) catalyze electrochemical O 2 , H + , and CO 2 reduction (14,(16)(17)(18)(19). Several excellent metalloporphyrin-based O 2 reduction electrocatalysts have been reported, some mimicking natural active sites and some inspired by their design (14,20). Of these catalysts, the Fe-based catalysts generally have provided valuable insights into the O 2 reduction mechanism and a deeper understanding of the structure-function correlations of key enzymes such as CcO, the terminal enzyme in the mitochondrial electron transport chain (1, 21). Similarly, several naturally occurring enzymes have been used as electrocatalysts for O 2 reduction (e.g., CcO, microperoxidase) and substrate oxidation using O 2 (e.g., cytochrome P450) (22-24). Apart from porphyrins, transition metal complexes of corroles, a related macrocycle that lacks one of the four mesocarbons of porphyrins, have been used extensively as electrocatalysts for O 2 reduction and H 2 O oxidation (5, 6). These catalytic systems are efficient in catalyzing selective O 2 activation/reduction reactions when the electrons (i.e., the reducing component) are provided from the electrode and O 2 (the oxidizing component) is obtai...

“…As determined from the recently published high-resolution x-ray structures from P. denitrificans (3) and the bovine-heart enzymes (4), possible candidates for L are (su I, P. denitrificans numbering is used): E278, Y280, T344, T351, K354, D399, H325 (which may be a CUB ligand), H276, H326 (the His ligands of CUB), and the propionate side chains of heme a3. Several of these groups have been proposed to be directly involved in proton pumping (3,4 [11] where A is the molar ionic specific conductances of the ions in units of f1-1'mol-1 dm2, [HPH] [13] The factor XB, introduced in Eq. 13, has a value of -1 or + 1 in buffers in which the unprotonated form is negative or neutral in charge, respectively.…”

Section: Discussionmentioning

confidence: 99%

“…7) has been used to investigate protonation reactions associated with reduction of dioxygen by partly or fully reduced cytochrome c oxidase (12,13). In this work, we have studied proton uptake and release in the binuclear center following laser-flash photolysis of the carbon monoxide-mixed valence (partly reduced) complex of cytochrome c oxidase in the absence of dioxygen.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Kinetic coupling between electron and proton transfer in cytochrome c oxidase: simultaneous measurements of conductance and absorbance changes.

Ädelroth

Sigurdson²,

Hallén³

et al. 1996

Bovine heart cytochrome c oxidase is an electron-current driven proton pump. To investigate the mechanism by which this pump operates it is important to study individual electron-and proton-transfer reactions in the enzyme, and key reactions in which they are kinetically and thermodynamically coupled. In this work, we have simultaneously measured absorbance changes associated with electron-transfer reactions and conductance changes associated with protonation reactions following pulsed illumination of the photolabile complex of partly reduced bovine cytochrome c oxidase and carbon monoxide. Following CO dissociation, several kinetic phases in the absorbance changes were observed with time constants ranging from -3 ,us to several milliseconds, reflecting internal electrontransfer reactions within the enzyme. The data show that the rate of one of these electron-transfer reactions, from cytochrome a3 to a on a millisecond time scale, is controlled by a proton-transfer reaction. These results are discussed in terms of a model in which cytochrome a3 interacts electrostatically with a protonatable group, L, in the vicinity of the binuclear center, in equilibrium with the bulk through a proton-conducting pathway, which determines the rate of proton transfer (and indirectly also of electron transfer). The interaction energy of cytochrome a3 with L was determined independently from the pH dependence of the extent of the millisecond-electron transfer and the number of protons released, as determined from the conductance measurements. The magnitude of the interaction energy, 70 meV (1 eV =