The terminal reaction cascade of water oxidation: Proton and oxygen release

In cyanobacteria, algae and plants Photosystem II produces the oxygen we breathe. Driven and clocked by light quanta, the catalytic Mn₄Ca-tyrosine centre accumulates four oxidising equivalents before it abstracts four electrons from water, liberating dioxygen and protons. Aiming at intermediates of the terminal four-electron cascade, we previously have suppressed this reaction by elevating the oxygen pressure, thereby stabilising one redox intermediate. Here, we established a similar suppression by increasing the proton concentration. Data were analysed in terms of only one (peroxy) redox intermediate between the fourfold oxidised Mn₄Ca-tyrosine centre and oxygen release. The surprising result was that the release into the bulk of one proton per dioxygen is linked to the first and rate-limiting electron transfer in the cascade rather than to the second which produces free oxygen. The penultimate intermediate might thus be conceived as a fully deprotonated peroxy-moiety.     

Oxygen pressurized X-ray crystallography: probing the dioxygen binding site in cofactorless urate oxidase and implications for its catalytic mechanism

The localization of dioxygen sites in oxygen-binding proteins is a nontrivial experimental task and is often suggested through indirect methods such as using xenon or halide anions as oxygen probes. In this study, a straightforward method based on x-ray crystallography under high pressure of pure oxygen has been developed. An application is given on urate oxidase (UOX), a cofactorless enzyme that catalyzes the oxidation of uric acid to 5-hydroxyisourate in the presence of dioxygen. UOX crystals in complex with a competitive inhibitor of its natural substrate are submitted to an increasing pressure of 1.0, 2.5, or 4.0 MPa of gaseous oxygen. The results clearly show that dioxygen binds within the active site at a location where a water molecule is usually observed but does not bind in the already characterized specific hydrophobic pocket of xenon. Moreover, crystallizing UOX in the presence of a large excess of chloride (NaCl) shows that one chloride ion goes at the same location as the oxygen. The dioxygen hydrophilic environment (an asparagine, a histidine, and a threonine residues), its absence within the xenon binding site, and its location identical to a water molecule or a chloride ion suggest that the dioxygen site is mainly polar. The implication of the dioxygen location on the mechanism is discussed with respect to the experimentally suggested transient intermediates during the reaction cascade.     

Structure of the bound dioxygen species in the cytochrome oxidase reaction of cytochrome cd1 nitrite reductase

Reduction of dioxygen to water is a key process in aerobic life, but atomic details of this reaction have been elusive because of difficulties in observing active oxygen intermediates by crystallography. Cytochrome cd(1) is a bifunctional enzyme, capable of catalyzing the one-electron reduction of nitrite to nitric oxide, and the four-electron reduction of dioxygen to water. The latter is a cytochrome oxidase reaction. Here we describe the structure of an active dioxygen species in the enzyme captured by cryo-trapping. The productive binding mode of dioxygen in the active site is very similar to that of nitrite and suggests that the catalytic mechanisms of oxygen reduction and nitrite reduction are closely related. This finding has implications to the understanding of the evolution of oxygen-reducing enzymes. Comparison of the dioxygen complex to complexes of cytochrome cd(1) with stable diatomic ligands shows that nitric oxide and cyanide bind in a similar bent conformation to the iron as dioxygen whereas carbon monoxide forms a linear complex. The significance of these differences is discussed.     

Dioxygen solubility in aqueous phosphatidylcholine dispersions

The solubility of molecular oxygen, or dioxygen, in low weight percent (1.5%) sonicated dimyristoylphosphatidylcholine (DMPC) aqueous dispersions saturated with air has been measured as a function of temperature between 10 degrees C and 40 degrees C. A modified Winkler technique was used involving a dual cell coulometric titration with voltammetric endpoint detection in a mixed solvent (methanol/water). The results indicate that dioxygen is approximately four times more soluble in the liquid crystalline bilayers (above 24 degrees C) than in the gel state bilayers (below 24 degrees C). The solubility of dioxygen in the bilayer does not appear to be strongly temperature dependent on either side of the 24 degrees C phase transition. The dioxygen solubility in gel state DMPC is approximately equal to that in water at the same temperature. Our result are contrasted with recent measurements made using EPR spin labels.     

Photosynthetic water oxidation at high O2 backpressure monitored by delayed chlorophyll fluorescence

The atmospheric dioxygen is produced by photosynthetic organisms. This light-driven process culminates in what appears as one step: a four-electron abstraction from two water molecules bound to the Mn4Ca complex of photosystem II. Recently, an intermediate of the O2-producing reaction sequence was stabilized by elevated oxygen backpressure and detected by UV flash photometry [Clausen, J., and Junge, W. (2004) Nature 430, 480]. We scrutinized its properties by delayed chlorophyll fluorescence measurements. Half-suppression of oxygen evolution was observed at a similar O2 pressure of 2.3 bar, as previously, now with photosystem II membrane particles from spinach, without artificial electron acceptors, and at a high signal-to-noise ratio. The data are tentatively interpreted as the stabilization of a 2-fold oxidized state of the catalytic center (S2*) with bound peroxide and its slow conversion into the normal S2 state by the release of peroxide.     

Primary intermediate in the reaction of mixed-valence cytochrome c oxidase with oxygen

The reaction of dioxygen with mixed-valence cytochrome c oxidase was followed in a rapid-mixing continuous-flow apparatus. The optical absorption difference spectrum and a kinetic analysis confirm the presence of the primary oxygen intermediate in the 0-100-microseconds time window. The resonance Raman spectrum of the iron-dioxygen stretching mode (568 cm-1) supplies evidence that the degree of electron transfer from the iron atom to the dioxygen is similar to that in oxy complexes of other heme proteins. Thus, the Fe-O2 bond does not display any unique structural features that could account for the rapid reduction of dioxygen to water. Furthermore, the frequency of the iron-dioxygen stretching mode is the same as that of the primary intermediate in the fully reduced enzyme, indicating that the oxidation state of cytochrome a plays no role in controlling the initial properties of the oxygen binding site.     

Search for intermediates of photosynthetic water oxidation

Photosystem II of cyanobacteria and plants incorporates the catalytic centre of water oxidation. Powered and clocked by quanta of light the centre accumulates four oxidising equivalents before oxygen is released. The first three oxidising equivalents are stored on the Mn₄Ca-cluster, raising its formal oxidation state from S₀ to S₃ and the third on Y_Z, producing S₃ Y_Z^ox. From there on water oxidation proceeds in what appears as a single reaction step (S₃ Y_Z^ox(H₂O)₂O₂ + 4H⁺ + S₀. Intermediate oxidation products of bound water had not been detected, until our recent report on the stabilisation of such an intermediate by high oxygen pressure (NATURE 430, 2004, 480–483). Based on the oxygen titration (half-point 2.3 bar) the standard free-energy profile of a reaction sequence with a single intermediate was calculated. It revealed a rather small difference (−3 kJ mol⁻¹) between the starting state [S₃Y_Z^OX and the product state S₀Y_Z + O₂ + 4H⁺ . Here we describe the tests for side effects of exposing core particles to high oxygen pressure. We found the reduction of P₆₈₀^{+ ·} in ns and the reduction/dismutation of quinones at the acceptor side of PSII both unaffected, and the inhibition of the oxygen evolving reaction by exposure to high O₂-pressure was fully reversible by decompression to atmospheric conditions.     

Cooperative binding of oxygen to the water-splitting enzyme in the filamentous cyanobacterium Oscillatoria chalybea

In the filamentous cyanobacterium Oscillatoria chalybea photolysis of water does not take place in the complete absence of oxygen. A catalytic oxygen partial pressure of 15x10(-6) Torr has to be present for effective water splitting to occur. By means of mass spectrometry we measured the photosynthetic oxygen evolution in the presence of H(2)(18)O in dependence on the oxygen partial pressure of the atmosphere and analysed the liberations of (16)O(2), (16)O(18)O and (18)O(2) simultaneously. The observed dependences of the light-induced oxygen evolution on bound oxygen yield sigmoidal curves. Hill coefficient values of 3.0, 3.1 and 3.2, respectively, suggest that the binding is cooperative and that four molecules of oxygen have to be bound per chain to the oxygen evolving complex. Oxygen seems to prime the water-splitting reaction by redox steering of the S-state system, putting it in the dark into the condition from which water splitting can start. It appears that in O. chalybea an interaction of oxygen with S(0) and S(1) leads to S(2) and S(3), thus yielding the typical oxygen evolution pattern in which even after extensive dark adaptation substantial amounts of Y(1) and Y(2) are found. The interacting oxygen is apparently reduced to hydrogen peroxide. Mass spectrometry permits to distinguish this highly specific oxygen requirement from the interaction of bulk atmospheric oxygen with the oxygen evolving complex of the cyanobacterium. This interaction leads to the formation H(2)O(2) which is decomposed under O(2) evolution in the light. The dependence on oxygen-partial pressure and temperature is analysed. Structural peculiarities of the cyanobacterial reaction centre of photosystem II referring to the extrinsic peptides might play a role.     

X-ray, ESR, and quantum mechanics studies unravel a spin well in the cofactor-less urate oxidase

Urate oxidase (EC 1.7.3.3 or UOX) catalyzes the conversion of uric acid using gaseous molecular oxygen to 5-hydroxyisourate and hydrogen peroxide in absence of any cofactor or transition metal. The catalytic mechanism was investigated using X-ray diffraction, electron spin resonance spectroscopy (ESR), and quantum mechanics calculations. The X-ray structure of the anaerobic enzyme-substrate complex gives credit to substrate activation before the dioxygen fixation in the peroxo hole, where incoming and outgoing reagents (dioxygen, water, and hydrogen peroxide molecules) are handled. ESR spectroscopy establishes the initial monoelectron activation of the substrate without the participation of dioxygen. In addition, both X-ray structure and quantum mechanic calculations promote a conserved base oxidative system as the main structural features in UOX that protonates/deprotonates and activate the substrate into the doublet state now able to satisfy the Wigner's spin selection rule for reaction with molecular oxygen in its triplet ground state.     

Eight steps preceding O-O bond formation in oxygenic photosynthesis--a basic reaction cycle of the Photosystem II manganese complex

In oxygenic photosynthesis, water is split at a Mn(4)Ca complex bound to the proteins of photosystem II (PSII). Powered by four quanta of visible light, four electrons and four protons are removed from two water molecules before dioxygen is released. By this process, water becomes an inexhaustible source of the protons and electrons needed for primary biomass formation. On the basis of structural and spectroscopic data, we recently have introduced a basic reaction cycle of water oxidation which extends the classical S-state cycle [B. Kok, B. Forbush, M. McGloin, Cooperation of charges in photosynthetic O2 evolution- I. A linear four-step mechanism, Photochem. Photobiol. 11 (1970) 457-475] by taking into account also the role and sequence of deprotonation events [H. Dau, M. Haumann, Reaction cycle of photosynthetic water oxidation in plants and cyanobacteria, Science 312 (2006) 1471-1472]. We propose that the outwardly convoluted and irregular events of the classical S-state cycle are governed by a simple underlying principle: protons and electrons are removed strictly alternately from the Mn complex. Starting in I(0), eight successive steps of alternate proton and electron removal lead to I(8) and only then the O-O bond is formed. Thus not only four oxidizing equivalents, but also four bases are accumulated prior to the onset of dioxygen formation. After reviewing the kinetic properties of the individual S-state transition, we show that the proposed basic model explains a large body of experimental results straightforwardly. Furthermore we discuss how the I-cycle model addresses the redox-potential problem of PSII water oxidation and we propose that the accumulated bases facilitate dioxygen formation by acting as proton acceptors.     

Photosystem II: The machinery of photosynthetic water splitting

This review summarizes our current state of knowledge on the structural organization and functional pattern of photosynthetic water splitting in the multimeric Photosystem II (PS II) complex, which acts as a light-driven water: plastoquinone-oxidoreductase. The overall process comprises three types of reaction sequences: (1) photon absorption and excited singlet state trapping by charge separation leading to the ion radical pair [Formula: see text] formation, (2) oxidative water splitting into four protons and molecular dioxygen at the water oxidizing complex (WOC) with P680+* as driving force and tyrosine Y(Z) as intermediary redox carrier, and (3) reduction of plastoquinone to plastoquinol at the special Q(B) binding site with Q(A)-* acting as reductant. Based on recent progress in structure analysis and using new theoretical approaches the mechanism of reaction sequence (1) is discussed with special emphasis on the excited energy transfer pathways and the sequence of charge transfer steps: [Formula: see text] where (1)(RC-PC)* denotes the excited singlet state (1)P680* of the reaction centre pigment complex. The structure of the catalytic Mn(4)O(X)Ca cluster of the WOC and the four step reaction sequence leading to oxidative water splitting are described and problems arising for the electronic configuration, in particular for the nature of redox state S(3), are discussed. The unravelling of the mode of O-O bond formation is of key relevance for understanding the mechanism of the process. This problem is not yet solved. A multistate model is proposed for S(3) and the functional role of proton shifts and hydrogen bond network(s) is emphasized. Analogously, the structure of the Q(B) site for PQ reduction to PQH(2) and the energetic and kinetics of the two step redox reaction sequence are described. Furthermore, the relevance of the protein dynamics and the role of water molecules for its flexibility are briefly outlined. We end this review by presenting future perspectives on the water oxidation process.     

Oxygen activation by cytochrome P450 monooxygenase

Unlike photosystem II (PSII) that catalyzes formation of the O-O bond, the cytochromes P450 (P450), members of a superfamily of hemoproteins, catalyze the scission of the O-O bond of dioxygen molecules and insert a single oxygen atom into unactivated hydrocarbons through a hydrogen abstraction-oxygen rebound mechanism. Hydroxylation of the unactivated hydrocarbons at physiological temperatures is vital for many cellar processes such as the biosynthesis of many endogenous compounds and the detoxification of xenobiotics in humans and plants. Even though it carries out the opposite of the water splitting reaction, P450 may share similarities to PSII in proton delivery networks, oxygen and water access channels, and consecutive electron transfer processes. In this article, we review recent advances in understanding the molecular mechanisms by which P450 activates dioxygen.     

The mechanism of cathode reduction of oxygen in a carbon carrier-laccase system

The influence of temperature, oxygen pressure and inhibitors of laccase on the dioxygen electroreduction reaction has been examined at different solution pH. On the basis of obtained data, a reaction mechanism including electron transfer from the enzyme active site to the oxygen molecule is suggested as the slow step.     

Kinetic studies on the reaction between Trametes villosa laccase and dioxygen

A laccase from the fungus Trametes villosa (TviL) was investigated in order to elucidate the reaction mechanism of the reduction of dioxygen to water performed by this blue multi-copper oxidase. The ability of TviL to activate dioxygen was studied by stopped-flow experiments and under steady-state conditions. In the stopped-flow experiments TviL was reduced with a small excess of 4-hydroxyphenylacetic acid and afterwards the re-oxidation process was monitored by stopped-flow techniques by mixing with excess dioxygen. The reaction between reduced TviL and dioxygen was studied in the temperature range 10-35 degrees Celsius and with the concentration of dioxygen between 30 and 240microM. The observed rate constant k(obs) is found to be linear dependent on the dioxygen concentration and the observed second-order rate constant for the re-oxidation of reduced TviL is, at 25 degrees Celsius, determined to be 1.14x10(6)M(-1)s(-1). The activation energy, E(a), is from the same data determined to be 22kJmol(-1). Oxidation of different phenols (4-hydroxyphenylacetic acid, 4-hydroxybenzoic acid, guaiacolsulfonic acid and hydroquinone) under steady state conditions was investigated at concentrations of dioxygen ranging from 60 to 250microM. This line of experiments showed that TviL follows a ping-pong mechanism, and an observed second-order rate constant for the reaction with dioxygen of 7.1x10(5)M(-1)s(-1) at 25 degrees Celsius was found with 4-hydroxyphenylacetic acid as reducing substrate. The two kinetic methods resulted in observed rate constants of equal magnitudes for the reaction with dioxygen, which suggests that the rate limiting step(s) is (are) included in both the reactions studied by the two different techniques.     

Time-resolved oxygen production by PSII: chasing chemical intermediates

Photosystem II (PSII) produces dioxygen from water in a four-stepped process, which is driven by four quanta of light and catalysed by a Mn-cluster and tyrosine Z. Oxygen is liberated during one step, coined S(3)=>S(0). Chemical intermediates on the way from reversibly bound water to dioxygen have not yet been tracked, however, a break in the Arrhenius plot of the oxygen-evolving step has been taken as evidence for its existence. We scrutinised the temperature dependence of (i) UV-absorption transients attributable to the reduction of the Mn-cluster and tyrosine Z by water, and (ii) polarographic transients attributable to the release of dioxygen. Using a centrifugatable and kinetically competent Pt-electrode, we observed no deviation from a linear Arrhenius plot of oxygen release in the temperature range from -2 to 32 degrees C, and hence no evidence, by this approach, for a sufficiently long-lived chemical intermediate. The half-rise times of oxygen release differed between Synechocystis WT* (at 20 degrees C: 1.35 ms) and a point mutant (D1-D61N: 13.1 ms), and the activation energies differed between species (Spinacia oleracea, 30 kJ/mol versus Synechocystis, 41 kJ/mol) and preparations (PSII membranes, 41 kJ/mol versus core complexes, 33 kJ/mol, Synechocystis). Correction for polarographic artefacts revealed, for the first time, a temperature-dependent lag-phase of the polarographic transient (duration at 20 degrees C: 0.45 ms, activation energy: 31 kJ/mol), which was indicative of a short-lived intermediate. It was, however, not apparent in the UV-transients. Thus the "intermediate" was probably newly formed and transiently bound oxygen.     

Mechanisms underlying dioxygen reduction in laccases. Structural and modelling studies focusing on proton transfer

Background  

Laccases are enzymes that couple the oxidation of substrates with the reduction of dioxygen to water. They are the simplest members of the multi-copper oxidases and contain at least two types of copper centres; a mononuclear T1 and a trinuclear that includes two T3 and one T2 copper ions. Substrate oxidation takes place at the mononuclear centre whereas reduction of oxygen to water occurs at the trinuclear centre.     

Eight steps preceding O-O bond formation in oxygenic photosynthesis—A basic reaction cycle of the Photosystem II manganese complex

In oxygenic photosynthesis, water is split at a Mn₄Ca complex bound to the proteins of photosystem II (PSII). Powered by four quanta of visible light, four electrons and four protons are removed from two water molecules before dioxygen is released. By this process, water becomes an inexhaustible source of the protons and electrons needed for primary biomass formation. On the basis of structural and spectroscopic data, we recently have introduced a basic reaction cycle of water oxidation which extends the classical S-state cycle [B. Kok, B. Forbush, M. McGloin, Cooperation of charges in photosynthetic O2 evolution- I. A linear four-step mechanism, Photochem. Photobiol. 11 (1970) 457-475] by taking into account also the role and sequence of deprotonation events [H. Dau, M. Haumann, Reaction cycle of photosynthetic water oxidation in plants and cyanobacteria, Science 312 (2006) 1471-1472]. We propose that the outwardly convoluted and irregular events of the classical S-state cycle are governed by a simple underlying principle: protons and electrons are removed strictly alternately from the Mn complex. Starting in I₀, eight successive steps of alternate proton and electron removal lead to I₈ and only then the O-O bond is formed. Thus not only four oxidizing equivalents, but also four bases are accumulated prior to the onset of dioxygen formation. After reviewing the kinetic properties of the individual S-state transition, we show that the proposed basic model explains a large body of experimental results straightforwardly. Furthermore we discuss how the I-cycle model addresses the redox-potential problem of PSII water oxidation and we propose that the accumulated bases facilitate dioxygen formation by acting as proton acceptors.     

Looking for the minimum common denominator in haem–copper oxygen reductases: Towards a unified catalytic mechanism

Haem–copper oxygen reductases are transmembrane protein complexes that reduce dioxygen to water and pump protons across the mitochondrial or periplasmatic membrane, contributing to the transmembrane difference of electrochemical potential. Seven years ago we proposed a classification of these enzymes into three different families (A, B and C), based on the amino acid residues of their proton channels and amino acid sequence comparison, later supported by the so far identified characteristics of the catalytic centre of members from each family. The three families have in common the same general structural fold of the catalytic subunit, which contains the same or analogous prosthetic groups, and proton channels. These observations raise the hypothesis that the mechanisms for dioxygen reduction, proton pumping and the coupling of the two processes may be the same for all these enzymes. Under this hypothesis, they should be performed and controlled by the same or equivalent elements/events, and the identification of retained elements in all families will reveal their importance and may prompt the definition of the enzyme operating mode. Thus, we believe that the search for a minimum common denominator has a crucial importance, and in this article we highlight what is already established for the haem–copper oxygen reductases and emphasize the main questions still unanswered in a comprehensive basis.     

Looking for the minimum common denominator in haem-copper oxygen reductases: towards a unified catalytic mechanism

Haem-copper oxygen reductases are transmembrane protein complexes that reduce dioxygen to water and pump protons across the mitochondrial or periplasmatic membrane, contributing to the transmembrane difference of electrochemical potential. Seven years ago we proposed a classification of these enzymes into three different families (A, B and C), based on the amino acid residues of their proton channels and amino acid sequence comparison, later supported by the so far identified characteristics of the catalytic centre of members from each family. The three families have in common the same general structural fold of the catalytic subunit, which contains the same or analogous prosthetic groups, and proton channels. These observations raise the hypothesis that the mechanisms for dioxygen reduction, proton pumping and the coupling of the two processes may be the same for all these enzymes. Under this hypothesis, they should be performed and controlled by the same or equivalent elements/events, and the identification of retained elements in all families will reveal their importance and may prompt the definition of the enzyme operating mode. Thus, we believe that the search for a minimum common denominator has a crucial importance, and in this article we highlight what is already established for the haem-copper oxygen reductases and emphasize the main questions still unanswered in a comprehensive basis.     

Reaction of ferrous cytochrome c peroxidase with dioxygen: site-directed mutagenesis provides evidence for rapid reduction of dioxygen by intramolecular electron transfer from the compound I radical site.

The reaction of dioxygen with the ferrous forms of the cloned cytochrome c peroxidase [CCP(MI)] and mutants of CCP(MI) prepared by site-directed mutagenesis was studied by photolysis of the respective ferrous-CO complexes in the presence of dioxygen. Reaction of ferrous CCP(MI) with dioxygen transiently formed a FeII-O2 complex (bimolecular rate constant = (3.8 +/- 0.3) x 10(4) M-1 s-1 at pH 6.0; 23 degrees C) that reacted further (first-order rate constant = 4 +/- 1 s-1) to form a product with an absorption spectrum and an EPR radical signal at g = 2.00 that were identical to those of compound I formed by the reaction of CCP(MI)III with peroxide. Thus, the product of the reaction of CCP(MI)II with dioxygen retained three of the four oxidizing equivalents of dioxygen. Gel electrophoresis of the CCP(MI)II + dioxygen reaction products showed that covalent dimeric and trimeric forms of CCP(MI) were produced by the reaction of CCP(MI)II with dioxygen. Photolysis of the CCP(MI)II-CO complex in the presence of ferrous cytochrome c prevented the appearance of the cross-linked forms and resulted in the oxidation of 3 mol of cytochrome c/mol of CCP(MI)II-CO added. The results provide evidence that reaction of CCP(MI)II with dioxygen causes transient oxidation of the enzyme by 1 equiv above the normal compound I oxidation state. Mutations that eliminate the broad EPR signal at g = 2.00 characteristic of the compound I radical also prevented the rapid oxidation of the ferrous enzyme by dioxygen.(ABSTRACT TRUNCATED AT 250 WORDS)