Crystallographic evidence for dioxygen interactions with iron proteins

The interaction of dioxygen with iron plays a key role in many important biological processes, such as dioxygen transport in the bloodstream and the reduction of dioxygen by iron in respiration. However, the catalytic mechanisms employed, for example in ligand oxidation, are not fully understood at the current time despite intensive biochemical, spectroscopic and structural studies. This review outlines the structural evidence obtained by X-ray crystallographic methods for the nature of the interactions between dioxygen and the metal in iron-containing proteins. Proteins involved in iron transport or electron transfer are not included.     

Laccases: Complex architectures for one-electron oxidations

Laccase (p-diphenol:dioxygen oxidoreductase), one of the earliest discovered enzymes, contains four copper ions in two active sites and catalyzes a one-electron oxidation of substrates such as phenols and their derivatives, or aromatic amines, coupled to a four-electron reduction of dioxygen to water. The catalytic mechanism has been studied for decades but is still not completely elucidated, especially in terms of the reduction of dioxygen to water. The key structural features of this enzyme are under investigation in several groups using techniques such as X-ray diffraction, electron paramagnetic resonance (EPR) spectroscopy, and site-directed mutagenesis. The high interest in laccases is explained by the large number of biotechnological applications. In this review, the most recent research on the overall structural features as well as on the structures and properties of the active sites are summarized, along with currently proposed mechanisms of reaction.     

Heme oxygenase, steering dioxygen activation toward heme hydroxylation

The activation of dioxygen by heme oxygenase proceeds via formation of an obligatory ferric hydroperoxide intermediate (FeIII-OOH), as is the case in the activation of dioxygen by monooxygenase enzymes. This review summarizes current understanding of the structural and dynamic properties in heme oxygenase that channel the reactivity of the FeIII-OOH intermediate toward heme hydroxylation rather than oxoferryl formation. In addition, structural and electronic factors dictating the regiospecificity of heme oxygenation are analyzed in the context of recent X-ray and NMR spectroscopic studies. Differences in mechanism between heme hydroxylation, as carried out by heme oxygenase, and the coupled oxidation process, are also addressed.     

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.     

Electrochemistry of 2-thio- and 2-oxo-pyrimidines in dimethyl formamide in the presence of dioxygen

Electrocatalysis of dioxygen reduction to superoxide ion in dimethylformamide is afforded by a reversible interaction with thiol or hydroxo forms of pyrimidine which takes place only on the mercury surface electrode even in the absence of transition metal ions. The interaction is revealed by a polarographic wave and by a voltammetric peak which can be recorded only in the presence of dioxygen in the pyrimidines solution. The polarographic wave and voltammetric peak are attributed to the reduction of the oxygenated pyrimidine. In the range of concentration where the polarographic limiting currents due to the reduction of the oxygenated and unoxygenated pyrimidine are of the same order of magnitude, the stoichiometric ratio dioxygen/pyrimidine is determined to be 1. The electrode process probably involves the formation of a complex of mercury with the thiol or the hydroxo form of the pyrimidine on the electrode surface. Adsorption or coadsorption of the reactants seems to be involved.     

The origin of the Fe(IV)=O intermediates in cytochrome aa(3) oxidase

The dioxygen reduction mechanism in cytochrome oxidases relies on proton control of the electron transfer events that drive the process. Proton delivery and proton channels in the protein that are relevant to substrate reduction and proton pumping are considered, and the current status of this area is summarized. We propose a mechanism in which the coupling of the oxygen reduction chemistry to proton translocation (P→F transition) is related to the properties of two groups of highly conserved residues, namely, His411/G386-T389 and the heme a(3)-propionateA-D399-H403 chain.     

Chemical and spectroscopic evidence for the formation of a ferryl Fea3 intermediate during turnover of cytochrome c oxidase

When partially reduced cytochrome c oxidase samples are reoxidized with dioxygen, an EPR-silent dioxygen intermediate, which is at the three-electron level of dioxygen reduction, is trapped at the dioxygen reduction site. The intermediate has novel spectral features at 580 and 537 nm. Combined optical and EPR results reveal that this intermediate reacts rapidly with CO at 277-298 K causing the abolition of the 580/537 mm features and the appearance of a rhombic CuB EPR signal. A ferryl Fea3, or an intermediate at the same formal level of oxidation, is proposed to oxidize CO to CO2 producing an EPR-detectable CuB adjacent to a low-spin ferrous Fea3-dioxygen (or carbon monoxide) adduct.     

Structural elements involved in electron-coupled proton transfer in cytochrome c oxidase

Haem-copper oxidases are the last components of the respiratory chains in aerobic organisms. These membrane-bound enzymes energetically couple the electron transfer (eT) reactions associated with reduction of dioxygen to water, to proton pumping across the membrane. Even though the mechanism of proton pumping at the molecular level still remains to be uncovered, recent progress has presented us with the structural features of the pumping machinery and detailed information about the eT and proton-transfer reactions associated with the pumping process.     

The origin of the FeIV = O intermediates in cytochrome aa3 oxidase

The dioxygen reduction mechanism in cytochrome oxidases relies on proton control of the electron transfer events that drive the process. Proton delivery and proton channels in the protein that are relevant to substrate reduction and proton pumping are considered, and the current status of this area is summarized. We propose a mechanism in which the coupling of the oxygen reduction chemistry to proton translocation (P → F transition) is related to the properties of two groups of highly conserved residues, namely, His411/G386-T389 and the heme a₃–propionateA–D399–H403 chain. This article is part of a Special Issue entitled: Respiratory Oxidases.     

Calorimetric studies of oxyhemoglobin dissociation. II. Erythrocytic oxygen depletion by sodium dithionite

Dithionite causes the depletion of dioxygen from suspensions of erythrocytes by reduction of the external dioxygen and not by diffusion into the cell. The molar enthalpy for the reduction shows a small difference with respect to the values found for free hemoglobin; and the normal stoichiometry of 2 moles dithionite/mole dioxygen found there is not observed with erythrocytes. At low hematocrit, the stoichiometry is 2.6:1 and decreases to 1.5:1 at high hematocrit. The change is not due to differences in the hemoglobin saturation or to an inability of dithionite to reduce all dioxygen present at the higher hematocrit. Neither catalase nor peroxidase added to the extracellular volume significantly alters the stoichiometry or the enthalpy of dioxygen reduction by dithionite. Addition of superoxide dismutase, however, restores the normal stoichiometry at high hematocrit and further increases the stoichiometry at low hematocrit. The calorimetrical signal of hydrogen peroxide, clearly seen with free dioxygen, is not present with erythrocytes. In all these cases the total heat evolved is the same.     

A new approach for studying fast biological reactions involving dioxygen: the reaction of fully reduced cytochrome c oxidase with O2

We describe a new method for studying rapid biological reactions involving dioxygen. This approach is based on the photolysis of a synthetic caged dioxygen carrier, which produces dioxygen on a fast time scale. The method was used to investigate the reduction of dioxygen to water by cytochrome c oxidase at room temperature following photolysis of a (mu-peroxo)(mu-hydroxo)bis[bis(bipyridyl)c obalt(III)] complex. The fact that dioxygen is generated in situ on a nanosecond or faster time scale avoids potential complications related to the fate of photodissociated CO in a conventional CO flow-flash experiment. The cobalt complex is stable at room temperature under anaerobic conditions and releases dioxygen upon irradiation at 355 nm with a quantum yield of 0.04. The complex does not react with reduced cytochrome oxidase or its reducing agents within the mixing time of the experiment, and its photoproducts do not interfere with the kinetics of the dioxygen reduction. The oxidation of the reduced cytochrome oxidase was monitored between 500 and 750 nm using a gated optical spectrometric multichannel analyzer following photodissociation of the cobalt complex. The data were analyzed using singular value decomposition and global exponential fitting, and two apparent lifetimes (380 +/- 50 micros and 1.7 +/- 0.2 ms) were resolved and compared to results from a conventional CO flow-flash experiment. The results show that approximately 90 microM dioxygen can be generated upon a single laser pulse and that this approach can be used to study other fast biological reactions involving O(2).     

The role of redox-active metals in the mechanism of action of bleomycin.

Belomycin is a glycopeptide antibiotic routinely used to treat human cancer. It is commonly thought to exert its biological effects as a metallodrug, which oxidatively damages DNA. This review systematically examines the properties of bleomycin which contribute to its reaction with DNA in vitro and may be important in the breakage of DNA in cells. Because strand cleavage results from the reductive activation of dioxygen by metallobleomycins, the mechanism of this process is given primary attention. Current understanding of the structures of the coordination sites of various metallobleomycins, their thermodynamic stabilities, their propensity to form adduct species, and their properties in ligand substitution reactions provide a foundation for consideration of the chemistry of dioxygen activation as well as a basis for thinking about the metal-speciation of bleomycin in biological systems. Oxidation-reduction pathways of iron-bleomycin, copper-bleomycin, and other metal-bleomycin species with O2 are then examined, including information on photochemical activation. With this background, structural and thermodynamic features of the binding interactions of DNA with bleomycin, its metal complexes, and adducts of metallobleomycins are reviewed. Then, the DNA cleavage reaction involving iron-bleomycin is scrutinized on the basis of the preceding discussion. Particular emphasis is placed on the constraints which the presence of DNA places on the mechanism of dioxygen activation. Similarly, the reactions of other metalloforms of bleomycin with DNA are reviewed. The last topic is an analysis of current understanding of the relationship of bleomycin-induced cellular DNA damage to the model developed above, which has evolved on the basis of chemical experimentation. Consideration is given to the question of the importance of DNA strand breakage caused by bleomycin for the mechanism of cytotoxic activity of the drug.     

Oxygen, oxidases, and the essential trace metals

The dominant function of dioxygen as the terminal electron acceptor in aerobic systems is well established; the roles of iron and copper in the terminal oxidases are less well understood. The minor, but crucial, part that dioxygen plays in other biological processes has recently attracted much attention. The chemistry of the reduction products of dioxygen is described and the possible relation of these products to the toxic properties of dioxygen is discussed. It is suggested that the uncontrolled reaction of dioxygen with reduced species, to give the superoxide ion, hydrogen peroxide, the hydroxyl radical and perhaps other entities derived from these, is potentially hazardous to the organism. Defences exist against these species, not least in the dismutases dependent on copper-zinc, manganese and iron, in catalase and in the selenium-dependent peroxidase. The effectiveness of these defences is examined and their reduction products of dioxygen during phagocytosis is discussed.     

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.     

Hyperoxidant states cause microbial cell differentiation by cell isolation from dioxygen

A general theory giving an explanation of microbial cell differentiation is presented. Based on experimental results, an unstable hyperoxidant state is postulated to trigger differentiation. Simple rules, involving the reduction of dioxygen and the isolation from dioxygen by diverse mechanisms, are proposed to govern transitions between the growth state and the differentiated states. With this view, common features of microbial differentiation processes, dimorphic growth, cell differentiation in dioxygen evolving phototrophs and in anaerobes are analyzed. The theory could have implications for understanding cell differentiation in higher organisms.     

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.     

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.     

Substrate binding and C-H bond activation in the soluble methane monooxygenase hydroxylase

 The selective oxidation of CH₄ to CH₃OH is a conceptually simple, yet functionally difficult, chemical transformation. In nature, this reaction is performed by methane monooxygenases, the soluble class of which employ carboxylate-bridged dinuclear iron centers to activate dioxygen. The process by which small molecules access the active site of the sMMO hydroxylase, the structures of intermediates in the catalytic reaction cycle, and mechanistic details about the attack on the C–H bond are subjects of intense investigation. In this commentary, we present our current views on exogenous ligand binding and dioxygen activation at the active site and the mechanism of alkane hydroxylation. Received: 20 October 1997 / Accepted: 26 February 1998     

Probing molecular structure of dioxygen reduction site of bacterial quinol oxidases through ligand binding to the redox metal centers

Cytochromes bo and bd are structurally unrelated terminal ubiquinol oxidases in the aerobic respiratory chain of Escherichia coli. The high-spin heme o-CuB binuclear center serves as the dioxygen reduction site for cytochrome bo, and the heme b595-heme d binuclear center for cytochrome bd. CuB coordinates three histidine ligands and serves as a transient ligand binding site en route to high-spin heme o one-electron donor to the oxy intermediate, and a binding site for bridging ligands like cyanide. In addition, it can protect the dioxygen reduction site through binding of a peroxide ion in the resting state, and connects directly or indirectly Tyr288 and Glu286 to carry out redox-driven proton pumping in the catalytic cycle. Contrary, heme b595 of cytochrome bd participate a similar role to CuB in ligand binding and dioxygen reduction but cannot perform such versatile roles because of its rigid structure.