Protonation reactions in relation to the coupling mechanism of bovine cytochrome c oxidase (original) (raw)
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The site of the redox-linked proton pump in eukaryotic cytochrome c oxidases
FEBS Letters, 1995
The electronic spectra of fully oxidized derivatives of some cytochrome c oxidase preparations are distinctly pH dependent. In general, the observed spectral shifts are greater in the case of pulsed derivatives compared to resting preparations and also, greater for preparations of the enzyme from shark skeletal muscle compared to beef heart. The low temperature near-infrared magnetic circular dichroism spectrum of the fully oxidized shark enzyme is not pH dependent in the experimental range, indicating the sensitivity of the visible region electronic spectrum to variation in pH to be due principally to changes at the heme a3-Cu B chromophore. The results are discussed in relation to proposed mechanisms of proton translocation in cytochrome c oxidase.
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2004
FTIR difference spectroscopy is used to reveal changes in the internal structure and amino acid protonation states of bovine cytochrome c oxidase (CcO) that occur upon photolysis of the CO adduct of the two-electron reduced (mixed valence, MV) and four-electron reduced (fully reduced, FR) forms of the enzyme. FTIR difference spectra were obtained in D 2 O (pH 6 -9.3) between the MV-CO adduct (heme a 3 and Cu B reduced; heme a and Cu A oxidized) and a photostationary state in which the MV-CO enzyme is photodissociated under constant illumination. In the photostationary state, part of the enzyme population has heme a 3 oxidized and heme a reduced. In MV-CO, the frequency of the stretch mode of CO bound to ferrous heme a 3 decreases from 1965.3 cm À 1 at pH* V 7 to 1963.7 cm À 1 at pH* 9.3. In the CO adduct of the fully reduced enzyme (FR-CO), the CO stretching frequency is observed at 1963.46 F 0.05 cm À 1 , independent of pH. This indicates that in MV-CO there is a group proximal to heme a that deprotonates with a pK a of about 8.3, but that remains protonated over the entire pH* range 6 -9.3 in FR-CO. The pK a of this group is therefore strongly coupled to the redox state of heme a. Following photodissociation of CO from heme a 3 in MV oxidases, the extent of electron transfer from heme a 3 to heme a shows a pH-dependent phase between pH 7 and 9, and a pH-independent phase at all pH's. The FTIR difference spectrum resulting from photolysis of MV-CO exhibits vibrational features of the protein backbone and side chains associated with (1) the loss of CO by the a 3 heme in the absence of electron transfer, (2) the pH-independent phase of the electron transfer, and (3) the pH-dependent phase of the electron transfer. Many infrared features change intensity or frequency during both electron transfer phases and thus appear as positive or negative features in the difference spectra. In particular, a negative band at 1735 cm À 1 and a positive band at 1412 cm À 1 are consistent with the deprotonation of the acidic residue E242. Positive features at 1552 and 1661 cm À 1 are due to amide backbone modes. Other positive and negative features between 1600 and 1700 cm À 1 are consistent with redoxinduced shifts in heme formyl vibrations, and the redox-linked protonation of an arginine residue, accompanying electron transfer from heme a 3 to heme a. An arginine could be the residue responsible for the pH-dependent shift in the carbonyl frequency of MV-CO. Specific possibilities as to the functional significance of these observations are discussed. D
Direct observation of protonation reactions during the catalytic cycle of cytochrome c oxidase
Proceedings of the National Academy of Sciences, 2003
Cytochrome c oxidase, the terminal protein in the respiratory chain, converts oxygen into water and helps generate the electrochemical gradient used in the synthesis of ATP. The catalytic action of cytochrome c oxidase involves electron transfer, proton transfer, and O 2 reduction. These events trigger specific molecular changes at the active site, which, in turn, influence changes throughout the protein, including alterations of amino acid side chain orientations, hydrogen bond patterns, and protonation states. We have used IR difference spectroscopy to investigate such modulations for the functional intermediate states E, R 2, Pm, and F. These spectra reveal deprotonation of its key glutamic acid E286 in the E and in the P m states. The consecutive deprotonation and reprotonation of E286 twice within one catalytic turnover illustrates the role of this residue as a proton shuttle. In addition, the spectra point toward deprotonation of a redox-active tyrosine, plausibly Y288, in the F intermediate. Structural insights into the molecular mechanism of catalysis based on the subtle molecular changes observed with IR difference spectroscopy are discussed. electron transfer ͉ infrared attenuated total reflection ͉ membrane protein ͉ bacteriorhodopsis ͉ glutamic acid
Radicals associated with the catalytic intermediates of bovine cytochrome c oxidase
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2002
Two radicals have been detected previously by electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopies in bovine cytochrome oxidase after reaction with hydrogen peroxide, but no correlation could be made with predicted levels of optically detectable intermediates (P M , F and F S) that are formed. This work has been extended by optical quantitation of intermediates in the EPR/ENDOR sample tubes, and by comparison with an analysis of intermediates formed by reaction with carbon monoxide in the presence of oxygen. The narrow radical, attributed previously to a porphyrin cation, is detectable at low levels even in untreated oxidase and increases with hydrogen peroxide treatments generally. It is presumed to arise from a side-reaction unrelated to the catalytic intermediates. The broad radical, attributed previously to a tryptophan radical, is observed only in samples with a significant level of F S but when F S is generated with hydrogen peroxide, is always accompanied by the narrow radical. When P M is produced at high pH with CO/O 2 , no EPR-detectable radicals are formed. Conversion of the CO/O 2-generated P M into F S when pH is lowered is accompanied by the appearance of a broad radical whose ENDOR spectrum corresponds to a tryptophan cation. Quantitation of its EPR intensity indicates that it is around 3% of the level of F S determined optically. It is concluded that low pH causes a change of protonation pattern in P M which induces partial electron redistribution and tryptophan cation radical formation in F S. These protonation changes may mimic a key step of the proton translocation process.
Biochemistry, 1996
A pH-dependent polarity change at the heme-copper binuclear center of the aa 3-type cytochrome c oxidase from Rhodobacter sphaeroides has been identified by low-temperature FTIR difference spectroscopy. "Light"-minus-"dark" FTIR difference spectra of the fully reduced CO-enzyme adduct were recorded at a range of pH, and the dominance of different populations of bound CO, R and , was found to vary with pH. An apparent pK a of about 7.3 for the transition was obtained. The R and forms are differentiated by different polarities at the heme-copper binuclear center of the enzyme, sensed by the stretching frequencies of CO bound either to the heme a 3 Fe or to Cu B. Several sitedirected mutants in the vicinity of the heme-copper center are shown to favor either the R or the forms of the enyzme, suggesting that what is being monitored is an equilibrium between two conformations of the reduced form of the oxidase. Recent resonance Raman evidence has been presented demonstrating that the R and forms of the R. sphaeroides oxidase exist at room temperature; therefore, the pH-dependent change in the polarity in the vicinity of the heme-copper center may be functionally significant.
Charge interactions of cytochrome c with cytochrome c oxidase
International Journal of Biochemistry, 1984
The pyridoxal phosphate (PLP) modification of the lysine amino groups in cytochrome c causes decrease in the reaction rate with cytochrome c oxidase. 2. The rate constants for (PLP),-cyt. c, PLP(Lys 86)-cyt. c, PLP(Lys 79)-cyt. c and native cytochrome c (at pH 7.4, 1=0.02) are 3.6 x IO-'sect', 5.5 x lo-'sect', 5.2 x IO-'set-' and 9.8 x 10-3secc', respectively. 3. In spite of the same positive charge of singly PLP-cytochromes c the reaction between PLP(Lys 86)-cyt. c and cyt. c oxidase exhibits the ionic strength dependence that differs from those of the PLP(Lys 79)-cyt. c. 4. The rate constants at zero and infinite ionic strength for PLP(Lys 86)-cyt. c is 2-fold less than that for PLP(Lys 79)-cyt. c. 5. The positively charged cytochrome c lysines 86 and 79 form two from four or five predicted complementary charge interactions with carboxyl groups on cytochrome c oxidase.
Radical formation in cytochrome c oxidase
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2011
The formation of radicals in bovine cytochrome c oxidase (bCcO), during the O 2 redox chemistry and proton translocation, is an unresolved controversial issue. To determine if radicals are formed in the catalytic reaction of bCcO under single turnover conditions, the reaction of O 2 with the enzyme, reduced by either ascorbate or dithionite, was initiated in a custom-built rapid freeze quenching (RFQ) device and the products were trapped at 77 K at reaction times ranging from 50 μs to 6 ms. Additional samples were hand mixed to attain multiple turnover conditions and quenched with a reaction time of minutes. X-band (9 GHz) continuous wave electron paramagnetic resonance (CW-EPR) spectra of the reaction products revealed the formation of a narrow radical with both reductants. D-band (130 GHz) pulsed EPR spectra allowed for the determination of the gtensor principal values and revealed that when ascorbate was used as the reductant the dominant radical species was localized on the ascorbyl moiety, and when dithionite was used as the reductant the radical was the SO 2 • − ion. When the contributions from the reductants are subtracted from the spectra, no evidence for a protein-based radical could be found in the reaction of O 2 with reduced bCcO. As a surrogate for radicals formed on reaction intermediates, the reaction of hydrogen peroxide (H 2 O 2) with oxidized bCcO was studied at pH 6 and pH 8 by trapping the products at 50 μs with the RFQ device to determine the initial reaction events. For comparison, radicals formed after several minutes of incubation were also examined, and X-band and D-band analysis led to the identification of radicals on Tyr-244 and Tyr-129. In the RFQ measurements, a peroxyl (R\O\O•) species was formed, presumably by the reaction between O 2 and an amino acid-based radical. It is postulated that Tyr-129 may play a central role as a proton loading site during proton translocation by ejecting a proton upon formation of the radical species and then becoming reprotonated during its reduction via a chain of three water molecules originating from the region of the propionate groups of heme a 3. This article is part of a Special Issue entitled: "Allosteric cooperativity in respiratory proteins".
The proton-pumping site of cytochrome c oxidase: a model of its structure and mechanism
Biochimica et biophysica acta, 1986
Cytochrome c oxidase is an electron-transfer driven proton pump. In this paper, we propose a complete chemical mechanism for the enzyme's proton-pumping site. The mechanism achieves pumping with chemical reaction steps localized at a redox center within the enzyme; no indirect coupling through protein conformational changes is required. The proposed mechanism is based on a novel redox-linked transition metal ligand substitution reaction. The use of this reaction leads in a straightforward manner to explicit mechanisms for achieving all of the processes previously determined (Blair, D.F., Gelles, J. and Chan, S.I. (1986) Biophys. J. 50, 713-733) to be needed to accomplish redox-linked proton pumping. These processes include: (1) modulation of the energetics of protonation/deprotonation reactions and modulation of the energetics of redox reactions by the structural state of the pumping site; (2) control of the rates of the pump's redox reactions with its electron-transfer part...
Proceedings of the National Academy of Sciences of the United States of America, 2015
Molecular oxygen acts as the terminal electron sink in the respiratory chains of aerobic organisms. Cytochrome c oxidase in the inner membrane of mitochondria and the plasma membrane of bacteria catalyzes the reduction of oxygen to water, and couples the free energy of the reaction to proton pumping across the membrane. The proton-pumping activity contributes to the proton electrochemical gradient, which drives the synthesis of ATP. Based on kinetic experiments on the O-O bond splitting transition of the catalytic cycle (A → PR), it has been proposed that the electron transfer to the binuclear iron-copper center of O2 reduction initiates the proton pump mechanism. This key electron transfer event is coupled to an internal proton transfer from a conserved glutamic acid to the proton-loading site of the pump. However, the proton may instead be transferred to the binuclear center to complete the oxygen reduction chemistry, which would constitute a short-circuit. Based on atomistic mole...