Catalytic Cycle Of Cytochrome Research Articles

Spectroscopic identification of the catalytic intermediates of cytochrome c oxidase in respiring heart mitochondria

The catalytic cycle of cytochrome c oxidase (COX) couples the reduction of oxygen to the translocation of protons across the inner mitochondrial membrane and involves several intermediate states of the heme a3-CuB binuclear center with distinct absorbance properties. The absorbance maximum close to 605 nm observed during respiration is commonly assigned to the fully reduced species of hemes a or a3 (R). However, by analyzing the absorbance of isolated enzyme and mitochondria in the Soret (420–450 nm), alpha (560–630 nm) and red (630–700 nm) spectral regions, we demonstrate that the Peroxy (P) and Ferryl (F) intermediates of the binuclear center are observed during respiration, while the R form is only detectable under nearly anoxic conditions in which electrons also accumulate in the higher extinction coefficient low spin a heme. This implies that a large fraction of COX (>50 %) is active, in contrast with assumptions that assign spectral changes only to R and/or reduced heme a. The concentration dependence of the COX chromophores and reduced c-type cytochromes on the transmembrane potential (ΔΨm) was determined in isolated mitochondria during substrate or apyrase titration to hydrolyze ATP. The cytochrome c-type redox levels indicated that soluble cytochrome c is out of equilibrium with respect to both Complex III and COX. Thermodynamic analyses confirmed that reactions involving the chromophores we assign as the P and F species of COX are ΔΨm-dependent, out of equilibrium, and therefore much slower than the ΔΨm-insensitive oxidation of the R intermediate, which is undetectable due to rapid oxygen binding.

Spectrophotometric and thermodynamic study on reaction of tetra (p-sulphnaphthyl) porphyrin Iron (II) complex with thiols and glycine ethyl ester as amodel for cytochrome (p-450) state c and haemochrome.

Studies using spectrophotometric titrations on dilute solution of tetra (p-sulphonaphthyl) porphinato Iron (II) [ TNPS4 Fe (II) ] in the presence of a large excess of thiols and glycine ethyl ester at high (PH=12.8) are reported. evidence for high spin five coordinate Iron (II) and low spin six coordinate Iron (II) complexes were found . Thermodynamic parameters and stability constants were also recorded , refer to exothermic reaction with negative values of ∆H and ∆G for both ligands thiols and glycine ethyl ester . LogKF , Log KD and (n) number of bounded ligands were calculated , to be found for (n=1-1.3) for thiol ligand and (n=1.8-2.1) for glycine ethyl ester ligand , which were assigned to five and six coordinate to the Iron (II) atom , respectively. These results are discussed in relation to the high spin Iron (II) state in the catalytic cycle of cytochrome (p-450) and to the low spin Iron (II) haemochrome.

Spectrophotometric study on reaction of non-naturally haem proteins with thiols in aqueous solutions .

Spectrophotometric titrations of dilute solution of tetra (p-sulphonaphthyl) porphinato Iron (II) [ TNPS4 Fe (II) ] in the presence of a large excess of thiols at high (PH=12.8) were studied. Evidence for high spin five coordinate iron (II) complexes were found . Thermodynamic parameters and stability constants , refer to exothermic reaction with negative values of ∆H and ∆G. LogKF , Log KD and (n) number of bounded ligands were calculated , and found to be (n=1-1.3) , which were assigned to five and six coordinate to the iron (II) atom. These results are discussed in relation to the high spin iron (II) state in the catalytic cycle of cytochrome (p-450)

Steps of the coupled charge translocation in the catalytic cycle of cytochrome c oxidase

Cytochromecoxidase (COX) terminates the respiratory chain in mitochondria and in plasmatic membrane of many aerobic bacteria. The enzyme reduces dioxygen molecule into water and the reaction is accompanied with generation of transmembrane difference of electric potentials. The energy conservation by COX is based on the vectorial organization of the chemical reaction due to substrate protons transfer from the negative phase into the active site to meet the electrons, coming from opposite side of the membrane. In addition, a half of free energy of redox-chemistry reaction is conserved through transmembrane proton pumping. Each of the reaction steps in the catalytic cycle of COX involves a sequence of coupled electron and proton transfer reaction. The time-resolved study of the coupled charge translocation during catalytic cycle of cytochromecoxidase is reviewed. Based on many fascinating parallels between the mechanisms of COX and pigment-protein complex of photosystem II from oxygenic photosynthetic organisms, the data described could be used in the light-driven water-splitting process.

The Protonation State of the Cross-linked Tyrosine during the Catalytic Cycle of Cytochrome c Oxidase

Cytochrome c oxidase is the terminal complex of the respiratory chain in mitochondria and some aerobic bacteria and is responsible for most of the O(2) consumption in biology. The key reaction in the catalysis of O(2) reduction is O-O bond scission that requires four electrons and a proton. In our recent work (Gorbikova, E. A., Belevich, I., Wikstrom, M., and Verkhovsky, M. I. (2008) Proc. Natl. Acad. Sci. U. S. A. 105, 10733-10737), it was shown that the cross-linked Tyr-280 (Paracoccus denitrificans numbering) provides the proton for O-O bond cleavage. The deprotonated Tyr-280 must be reprotonated later on in the catalytic cycle to serve as a proton donor for the next oxygen reduction event. To find the reaction step at which the cross-linked Tyr-280 becomes reprotonated, all further steps of the catalytic cycle after O-O bond cleavage were followed by infrared spectroscopy. We found that complete reprotonation of the tyrosine is linked to the formation of the one-electron reduced state coupled to reduction of the Cu(B) site.

S13.16 The role of the cross-linked Tyr in the catalytic cycle of cytochrome c oxidase

S13.16 The role of the cross-linked Tyr in the catalytic cycle of cytochrome c oxidase

Calculated Reaction Cycle of CytochromecOxidase

The catalytic cycle of cytochrome c oxidase has been simulated by means of quantum mechanical calculations. The experimental energetics of the catalytic cycle is nearly reproduced. The atomic structures of the intermediates are suggested. In particular, the structures of nonactive "resting" intermediates are proposed.

Valence Tautomerism and Coordinative Lability in Copper(II)−Imidazolyl−Semiquinonate Anion Radical Models for the CuB Center in Cytochrome c Oxidases

In contrast to imidazolyl−phenoxyl radical models for the putative histidinyl−tyrosyl radical intermediate in the catalytic cycle of cytochrome c oxidase (CcO), imidazolyl−semiquinone radicals are stable and may be quantitatively produced. Internal electron transfer (valence tautomerism) within a CuII−imidazolyl−hydroquinonate species formed upon deprotonation of the parent CuII−imidazolyl−hydroquinone complex results in a substitutionally labile CuI−imidazolyl−semiquinone radical system. The results show the potential reactivity of a (transient) CuBII−histidinyl−tyrosyl anion intermediate in the catalytic cycle of CcO.

Formamide probes a role for water in the catalytic cycle of cytochrome c oxidase

Formamide is a slow-onset inhibitor of mitochondrial cytochrome c oxidase that is proposed to act by blocking water movement through the protein. In the presence of formamide the redox level of mitochondrial cytochrome c oxidase evolves over the steady state as the apparent electron transfer rate from cytochrome a to cytochrome a 3 slows. At maximal inhibition cytochrome a and cytochrome c are fully reduced, whereas cytochrome a 3 and Cu B remain fully oxidized consistent with the idea that formamide interferes with electron transfer between cytochrome a and the oxygen reaction site. However, transient kinetic studies show that intrinsic rates of electron transfer are unchanged in the formamide-inhibited enzyme. Formamide inhibition is demonstrated for another member of the heme-oxidase family, cytochrome c oxidase from Bacillus subtilis, but the onset of inhibition is much quicker than for mitochondrial oxidase. If formamide inhibition arises from a steric blockade of water exchange during catalysis then water exchange in the smaller bacterial oxidase is more open. Subunit III removal from the mitochondrial oxidase hastens the onset of formamide inhibition suggesting a role for subunit III in controlling water exchange during the cytochrome c oxidase reaction.

Proton pumping mechanism and catalytic cycle of cytochrome c oxidase: Coulomb pump model with kinetic gating

Proton pumping mechanism and catalytic cycle of cytochrome c oxidase: Coulomb pump model with kinetic gating

Proton pumping mechanism and catalytic cycle of cytochrome c oxidase: Coulomb pump model with kinetic gating

Using electrostatic calculations, we have examined the dependence of the protonation state of cytochrome c oxidase from bovine heart on its redox state. Based on these calculations, we propose a possible scheme of redox-linked proton pumping. The scheme involves His291 – one of the ligands of the Cu B redox center – which plays the role of the proton loading site (PLS) of the pump. The mechanism of pumping is based on ET reaction between two hemes of the enzyme, which is coupled to a transfer of two protons. Upon ET, the first proton (fast reaction) is transferred to the PLS (His291), while subsequent transfer of the second “chemical” proton to the binuclear center (slow reaction) is accompanied by the ejection of the first (pumped) proton. Within the proposed model, we discuss the catalytic cycle of the enzyme.

The catalytic cycle of cytochrome c oxidase is not the sum of its two halves.

Membrane-bound cytochrome c oxidase catalyzes cell respiration in aerobic organisms and is a primary energy transducer in biology. The two halves of the catalytic cycle may be studied separately: in an oxidative phase, the enzyme is oxidized by O(2), and in a reductive phase, the oxidized enzyme is reduced before binding the next O(2) molecule. Here we show by time-resolved membrane potential and pH measurements with cytochrome oxidase liposomes that, with both phases in succession, two protons are translocated during each phase, one during each individual electron transfer step. However, when the reductive phase is not immediately preceded by oxidation, it follows a different reaction pathway no longer coupled to proton pumping. Metastable states with altered redox properties of the metal centers are accessed during turnover and relax when external electron donors are exhausted but recover after enzyme reduction and reoxidation by O(2). The efficiency of ATP synthesis might be regulated by switching between the two catalytic pathways.

Direct observation of protonation reactions during the catalytic cycle of cytochrome c oxidase.

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 O2 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, R2,Pm, and F. These spectra reveal deprotonation of its key glutamic acid E286 in the E and in the Pm 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.

The influence of temperature and osmolyte on the catalytic cycle of cytochrome c oxidase.

The influence of temperature on cytochrome c oxidase (CCO) catalytic activity was studied in the temperature range 240-308 K. Temperatures below 273 K required the inclusion of the osmolyte ethylene glycol. For steady-state activity between 278 and 308 K the activation energy was 12 kcal x mol-1; the molecular activity or turnover number was 12 s-1 at 280 K in the absence of ethylene glycol. CCO activity was studied between 240 and 277 K in the presence of ethylene glycol. The activation energy was 30 kcal x mol-1; the molecular activity was 1 s-1 at 280 K. Ethylene glycol inhibits CCO by lowering the activity of water. The rate limitation in electron transfer (ET) was not associated with ET into the CCO as cytochrome a was predominantly reduced in the aerobic steady state. The activity of CCO in flash-induced oxidation experiments was studied in the low temperature range in the presence of ethylene glycol. Flash photolysis of the reduced CO complex in the presence of oxygen resulted in three discernable processes. At 273 K the rate constants were 1500 s-1, 150 s-1 and 30 s-1 and these dropped to 220 s-1, 27 s-1 and 3 s-1 at 240 K. The activation energies were 5 kcal.mol-1, 7 kcal.mol-1, and 8 kcal.mol-1, respectively. The fastest rate we ascribe to the oxidation of cytochrome a3, the intermediate rate to cytochrome a oxidation and the slowest rate to the re-reduction of cytochrome a followed by its oxidation. There are two comparisons that are important: (a). with vs. without ethylene glycol and (b). steady state vs. flash-induced oxidation. When one makes these two comparisons it is clear that the CCO only senses the presence of osmolyte during the reductive portion of the catalytic cycle. In the present work that would mean after a flash-induced oxidation and the start of the next reduction/oxidation cycle.

Proton translocation by cytochrome c oxidase: a rejoinder to recent criticism.

Ten years ago, intermediate reaction steps in the catalytic cycle of cytochrome c oxidase were titrated with phosphorylation potential in isolated mitochondria, and the results were interpreted as evidence for thermodynamic linkage of proton translocation exclusively to the oxidative reaction steps of the catalytic cycle [Wikström, M. (1989) Nature 338, 776-778]. Michel has recently argued that this work was flawed, and proposed a mechanism in which one of the four steps of proton translocation is linked to the reductive phase of the catalytic cycle [Michel, H. (1999) Biochemistry 38, 15129-15140]. Here, the original data are scrutinized and related to information that has accumulated since this work was published. The analysis shows that the main conclusions from this work still hold. Michel's mechanism of proton translocation is briefly discussed, and found to be at odds with some experimental observations.

Preparation and Characterization of Oxoiron(IV) Chlorin Complexes as the First Models for a Reaction Intermediate in the Catalytic Cycle of Cytochrome d

As models for a reaction intermediate in the catalytic cycle of cytochrome d, two types of oxoferryl chlorin complexes, (TPC)Fe IV O(N-MeIm) and (TPC)Fe IV O (TPC=tetraphenylchlorin, N-MeIm=N-methylimidazole), have been prepared for the first time by an autoxidation reaction of (TPC)Fe II with O 2 . Oxyiron(II)(TPC) and μ-peroxo-bridged iron(III) dimer, (TPC)Fe III OOFe III (TPC), are also detected in the course of the reaction. The six-coordinated (TPC)Fe IV O(N-MeIm) complex is produced by adding N-methylimidazole to the μ-peroxo-bridged iron(III) chlorin dimer at -80 o C

Spectroscopic characterization of peroxo-iron(III) chlorin complexes. The first model for a reaction intermediate of cytochrome d

As reaction intermediate proposed in the catalytic cycle of cytochrome d, peroxo-iron(III) chlorin complexes are formed and characterized by using synthetic models. The peroxo complexes have been successfully formed by the reaction of chloro-iron(III) complexes of tetramesitylchlorin (H 2 TMC) and octaethylchlorin (H 2 OEC) with 2 equiv of the superoxide anion in acetonitrile under an argon atmosphere. The peroxo complexes of iron(III) chlorins afford characteristic electronic absorption spectra, in which the Soret band and the characteristic band for chlorin complex are largely red-shifted, as compared with those of the parent ferric chlorin complexes

ChemInform Abstract: Synthetic Porphyrins/Metalloporphyrins Which Mimic States in Catalytic Cycle of Cytochrome P‐450 and Peroxidases

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Is the internal electron transfer the rate-limiting step in the catalytic cycle of cytochrome c oxidase?

Is the internal electron transfer the rate-limiting step in the catalytic cycle of cytochrome c oxidase?

Resonance raman studies of models for the reduced states of cytochrome P 450

The catalytic cycle of cytochrome P 450 includes four stable states: a low spin ferric resting state A, a substrate bound high spin ferric state B, a high spin ferrous state C and a low spin ferrous oxy state D. When the system in the state C is exposed to carbon monoxide instead of oxygen a low spin ferrous carboxy state D′ is generated. Several models have been proposed for states B, C, D′ and recently state D [1]. A large variety of spectroscopic techniques have been used to probe the similarities between the model compounds and the actual enzymatic states. Resonance Raman spectroscopy (RR) is a very sensitive technique to investigate specifically the active site of hemoproteins. RR spectra have been obtained for Cyt P 450 from various origins [2–4]: when compared to those of other hemoproteins, the frequencies of the so-called ‘oxidation state’ marker band [5] are unusually low in states C and D′; ‘spin marker bands’ frequencies [5] have been used to monitor the coordination of the iron atom in the ferric states A and B. Recently the FeS stretching mode has been detected at 351 cm −1 in oxidized Cyt P 450 CAM [6]. We report here the results of a RR study of [Fe II(T pivPP)(X −)(L)]18C6Na +* complexes, as models for states C, D′ and D (an investigation of a state A model has already been published [7]). Using the nomenclature propsed in [8], the main RR frequencies are given in Table 1. t001 RR Frequencies (cm −1) of [Fe II(T pivPP)(X −)(L)]Na +18C6 Complexes. Complex Porphyrin vibration FeL vibr. X − L A B C D C 6HF 4S − 1341 a a a 369 Cl − 1343 1355 1494 1545 369 OH − 1344 1355 a a 371 C 6HF 4O − 1343 1354 a 1545 369 C 6HF 4S − CO 1364 a 1567 380 479 C 6HF 4S − O 2 1366 a a 379 a Not observed All the pentacoordinated ferrous species exhibit very similar porphyrinic frequencies. They compare well with the frequencies of the typical high spin ferrous complex Fe(TPP)(2-Me Im) (A = 1345, B = 1361, C = 1500 and D = 1538 [8]). Moreover the A frequency of the carboxy adduct is very close to that of Fe(TPP)(py)(CO) [12], whereas that of the oxy adduct is the same as that of Fe(T pivPP)(1-Me Im)(O 2) [9]. Therefore our RR data do not stress any special π donor properties of the RS − ligand that would induce an extra lowering of the oxidation state marker band frequencies. Soret excitation of the low frequency RR spectrum is readily accessible for the carboxy adduct λ max Soret 448 nm, λ exc 454,5 nm: it reveals a new strong polarized band at 479 cm −1. The intensity of this band decreases with partial photodissociation of the CO ligand. An isotopic substitution experiment, using 13CO, induces a 5 cm −1 lowering of its frequency. This is in good agreement with a calculated shift of −5 cm −1 for the stretching vibration of the FeCO moiety, using the harmonic oscillator approximation. This leads to the assignment of this band to the FeCO stretching vibration. This value is to be compared to those observed for MbCO, HbCO [10] and P 450CO (work in progress).