Kinetic studies of the reactions of O2 and NO with reduced Thermus thermophilus ba3 and bovine aa3 using photolabile carriers

The reactions of molecular oxygen (O₂) and nitric oxide (NO) with reduced Thermus thermophilus (Tt) ba₃ and bovine heart aa₃ were investigated by time-resolved optical absorption spectroscopy to establish possible relationships between the structural diversity of these enzymes and their reaction dynamics. To determine whether the photodissociated carbon monoxide (CO) in the CO flow-flash experiment affects the ligand binding dynamics, we monitored the reactions in the absence and presence of CO using photolabile O₂ and NO complexes. The binding of O₂/NO to reduced ba₃ in the absence of CO occurs with a second-order rate constant of 1 × 10⁹ M^? 1 s^? 1. This rate is 10-times faster than for the mammalian enzyme, and which is attributed to structural differences in the ligand channels of the two enzymes. Moreover, the O₂/NO binding in ba₃ is 10-times slower in the presence of the photodissociated CO while the rates are the same for the bovine enzyme. This indicates that the photodissociated CO directly or indirectly impedes O₂ and NO access to the active site in Tt ba₃, and that traditional CO flow-flash experiments do not accurately reflect the O₂ and NO binding kinetics in ba₃. We suggest that in ba₃ the binding of O₂ (NO) to heme a₃^2 + causes rapid dissociation of CO from Cu_B⁺ through steric or electronic effects or, alternatively, that the photodissociated CO does not bind to Cu_B⁺. These findings indicate that structural differences between Tt ba₃ and the bovine aa₃ enzyme are tightly linked to mechanistic differences in the functions of these enzymes. This article is part of a Special Issue entitled: Respiratory Oxidases.     

The rate-limiting step in O2 reduction by cytochrome ba3 from Thermus thermophilus

Cytochrome ba₃ (ba₃) of Thermus thermophilus (T. thermophilus) is a member of the heme–copper oxidase family, which has a binuclear catalytic center comprised of a heme (heme a₃) and a copper (Cu_B). The heme–copper oxidases generally catalyze the four electron reduction of molecular oxygen in a sequence involving several intermediates. We have investigated the reaction of the fully reduced ba₃ with O₂ using stopped-flow techniques. Transient visible absorption spectra indicated that a fraction of the enzyme decayed to the oxidized state within the dead time (~ 1 ms) of the stopped-flow instrument, while the remaining amount was in a reduced state that decayed slowly (k = 400 s^? 1) to the oxidized state without accumulation of detectable intermediates. Furthermore, no accumulation of intermediate species at 1 ms was detected in time resolved resonance Raman measurements of the reaction. These findings suggest that O₂ binds rapidly to heme a₃ in one fraction of the enzyme and progresses to the oxidized state. In the other fraction of the enzyme, O₂ binds transiently to a trap, likely Cu_B, prior to its migration to heme a₃ for the oxidative reaction, highlighting the critical role of Cu_B in regulating the oxygen reaction kinetics in the oxidase superfamily. This article is part of a Special Issue entitled: Respiratory Oxidases.     

High resolution structure of the ba3 cytochrome c oxidase from Thermus thermophilus in a lipidic environment

The fundamental chemistry underpinning aerobic life on Earth involves reduction of dioxygen to water with concomitant proton translocation. This process is catalyzed by members of the heme-copper oxidase (HCO) superfamily. Despite the availability of crystal structures for all types of HCO, the mode of action for this enzyme is not understood at the atomic level, namely how vectorial H⁺ and e^- transport are coupled. Toward addressing this problem, we report wild type and A120F mutant structures of the ba₃-type cytochrome c oxidase from Thermus thermophilus at 1.8 Å resolution. The enzyme has been crystallized from the lipidic cubic phase, which mimics the biological membrane environment. The structures reveal 20 ordered lipid molecules that occupy binding sites on the protein surface or mediate crystal packing interfaces. The interior of the protein encloses 53 water molecules, including 3 trapped in the designated K-path of proton transfer and 8 in a cluster seen also in A-type enzymes that likely functions in egress of product water and proton translocation. The hydrophobic O₂-uptake channel, connecting the active site to the lipid bilayer, contains a single water molecule nearest the Cu_B atom but otherwise exhibits no residual electron density. The active site contains strong electron density for a pair of bonded atoms bridging the heme Fe_a3 and Cu_B atoms that is best modeled as peroxide. The structure of ba₃-oxidase reveals new information about the positioning of the enzyme within the membrane and the nature of its interactions with lipid molecules. The atomic resolution details provide insight into the mechanisms of electron transfer, oxygen diffusion into the active site, reduction of oxygen to water, and pumping of protons across the membrane. The development of a robust system for production of ba₃-oxidase crystals diffracting to high resolution, together with an established expression system for generating mutants, opens the door for systematic structure-function studies.     

Kinetic studies of the reactions of O(2) and NO with reduced Thermus thermophilus ba(3) and bovine aa(3) using photolabile carriers

The reactions of molecular oxygen (O(2)) and nitric oxide (NO) with reduced Thermus thermophilus (Tt) ba(3) and bovine heart aa(3) were investigated by time-resolved optical absorption spectroscopy to establish possible relationships between the structural diversity of these enzymes and their reaction dynamics. To determine whether the photodissociated carbon monoxide (CO) in the CO flow-flash experiment affects the ligand binding dynamics, we monitored the reactions in the absence and presence of CO using photolabile O(2) and NO complexes. The binding of O(2)/NO to reduced ba(3) in the absence of CO occurs with a second-order rate constant of 1×10(9)M(-1)s(-1). This rate is 10-times faster than for the mammalian enzyme, and which is attributed to structural differences in the ligand channels of the two enzymes. Moreover, the O(2)/NO binding in ba(3) is 10-times slower in the presence of the photodissociated CO while the rates are the same for the bovine enzyme. This indicates that the photodissociated CO directly or indirectly impedes O(2) and NO access to the active site in Tt ba(3), and that traditional CO flow-flash experiments do not accurately reflect the O(2) and NO binding kinetics in ba(3). We suggest that in ba(3) the binding of O(2) (NO) to heme a(3)(2+) causes rapid dissociation of CO from Cu(B)(+) through steric or electronic effects or, alternatively, that the photodissociated CO does not bind to Cu(B)(+). These findings indicate that structural differences between Tt ba(3) and the bovine aa(3) enzyme are tightly linked to mechanistic differences in the functions of these enzymes. This article is part of a Special Issue entitled: Respiratory Oxidases.     

Spectroscopic and Kinetic Investigation of the Fully Reduced and Mixed Valence States of ba3-Cytochrome c Oxidase from Thermus thermophilus: A FOURIER TRANSFORM INFRARED (FTIR) AND TIME-RESOLVED STEP-SCAN FTIR STUDY*

The complete understanding of a molecular mechanism of action requires the thermodynamic and kinetic characterization of different states and intermediates. Cytochrome c oxidase reduces O₂ to H₂O, a reaction coupled to proton translocation across the membrane. Therefore, it is necessary to undertake a thorough characterization of the reduced form of the enzyme and the determination of the electron transfer processes and pathways between the redox-active centers. In this study Fourier transform infrared (FTIR) and time-resolved step-scan FTIR spectroscopy have been applied to study the fully reduced and mixed valence states of cytochrome ba₃ from Thermus thermophilus. We used as probe carbon monoxide (CO) to characterize both thermodynamically and kinetically the cytochrome ba₃-CO complex in the 5.25–10.10 pH/pD range and to study the reverse intramolecular electron transfer initiated by the photolysis of CO in the two-electron reduced form. The time-resolved step-scan FTIR data revealed no pH/pD dependence in both the decay of the transient Cu_B¹⁺-CO complex and rebinding to heme a₃ rates, suggesting that no structural change takes place in the vicinity of the binuclear center. Surprisingly, photodissociation of CO from the mixed valence form of the enzyme does not lead to reverse electron transfer from the reduced heme a₃ to the oxidized low-spin heme b, as observed in all the other aa₃ and bo₃ oxidases previously examined. The heme b-heme a₃ electron transfer is guaranteed, and therefore, there is no need for structural rearrangements and complex synchronized cooperativities. Comparison among the available structures of ba₃- and aa₃-cytochrome c oxidases identifies possible active pathways involved in the electron transfer processes and key structural elements that contribute to the different behavior observed in cytochrome ba₃.     

Structural changes that occur upon photolysis of the Fe(II)a3–CO complex in the cytochrome ba3-oxidase of Thermus thermophilus: A combined X-ray crystallographic and infrared spectral study demonstrates CO binding to CuB

The purpose of the work was to provide a crystallographic demonstration of the venerable idea that CO photolyzed from ferrous heme-a₃ moves to the nearby cuprous ion in the cytochrome c oxidases. Crystal structures of CO-bound cytochrome ba₃-oxidase from Thermus thermophilus, determined at ~ 2.8–3.2 Å resolution, reveal a Fe–C distance of ~ 2.0 Å, a Cu–O distance of 2.4 Å and a Fe–C–O angle of ~ 126°. Upon photodissociation at 100 K, X-ray structures indicate loss of Fe_a3–CO and appearance of Cu_B–CO having a Cu–C distance of ~ 1.9 Å and an O–Fe distance of ~ 2.3 Å. Absolute FTIR spectra recorded from single crystals of reduced ba₃–CO that had not been exposed to X-ray radiation, showed several peaks around 1975 cm^? 1; after photolysis at 100 K, the absolute FTIR spectra also showed a significant peak at 2050 cm^? 1. Analysis of the ‘light’ minus ‘dark’ difference spectra showed four very sharp CO stretching bands at 1970 cm^? 1, 1977 cm^? 1, 1981 cm^? 1, and 1985 cm^? 1, previously assigned to the Fe_a3–CO complex, and a significantly broader CO stretching band centered at ~ 2050 cm^? 1, previously assigned to the CO stretching frequency of Cu_B bound CO. As expected for light propagating along the tetragonal axis of the P4₃2₁2 space group, the single crystal spectra exhibit negligible dichroism. Absolute FTIR spectrometry of a CO-laden ba₃ crystal, exposed to an amount of X-ray radiation required to obtain structural data sets before FTIR characterization, showed a significant signal due to photogenerated CO₂ at 2337 cm^? 1 and one from traces of CO at 2133 cm^? 1; while bands associated with CO bound to either Fe_a3 or to Cu_B in “light” minus “dark” FTIR difference spectra shifted and broadened in response to X-ray exposure. In spite of considerable radiation damage to the crystals, both X-ray analysis at 2.8 and 3.2 Å and FTIR spectra support the long-held position that photolysis of Fe_a3–CO in cytochrome c oxidases leads to significant trapping of the CO on the Cu_B atom; Fe_a3 and Cu_B ligation, at the resolutions reported here, are otherwise unaltered. This article is part of a Special Issue entitled: Respiratory Oxidases.     

The copper-molybdenum antagonism in ruminants. III. Reaction of copper(II) with tetrathiomolybdate(VI)

The stoichiometry of the reaction between Cu(II) and MoS₄²⁻ in neutral aqueous solution was observed to proceed with a 1.5:1 Cu:Mo ratio. The reaction results in the reduction of Cu(II) and the quantitative formation of an insoluble solid. The results contrast with an earlier report of a 1:1 stoichiometric ratio, this latter ratio was, however, observed with Cu^II(albumin) as reactant, in this case no precipitate was observed. The insoluble products were examined by a number of spectroscopic techniques and by X-ray power diffraction and elemental analysis. Two products were identified. Solid A is isostructural with the known (NH₄)Cu^IMoV^VIS₄, i.e. has the composition M^ICuMoS₄, M^I= NH₄⁺, Na⁺ or Et₄N⁺. Solid B has the approximate composition CuMoS₄O_x, x=2−3 and contains Cu(I) and Mo(V) centres. Formation of compound B therefore involves an unusual internal two-electron redox process. The reaction and products are of particular biological significance.     

Concerted involvement of cooperative proton–electron linkage and water production in the proton pump of cytochrome c oxidase

In cytochrome c oxidase, oxido-reductions of heme a/Cu_A and heme a₃/Cu_B are cooperatively linked to proton transfer at acid/base groups in the enzyme. H⁺/e⁻ cooperative linkage at Fe_a3/Cu_B is envisaged to be involved in proton pump mechanisms confined to the binuclear center. Models have also been proposed which involve a role in proton pumping of cooperative H⁺/e⁻ linkage at heme a (and Cu_A). Observations will be presented on: (i) proton consumption in the reduction of molecular oxygen to H₂O in soluble bovine heart cytochrome c oxidase; (ii) proton release/uptake associated with anaerobic oxidation/reduction of heme a/Cu_A and heme a₃/Cu_B in the soluble oxidase; (iii) H⁺ release in the external phase (i.e. H⁺ pumping) associated with the oxidative (R → O transition), reductive (O → R transition) and a full catalytic cycle (R → O → R transition) of membrane-reconstituted cytochrome c oxidase. A model is presented in which cooperative H⁺/e⁻ linkage at heme a/Cu_A and heme a₃/Cu_B with acid/base clusters, C₁ and C₂ respectively, and protonmotive steps of the reduction of O₂ to water are involved in proton pumping.     

Reaction of nitric oxide with the oxidized di-heme and heme–copper oxygen-reducing centers of terminal oxidases: Different reaction pathways and end-products

Inhibition of terminal oxidases by nitric oxide (NO) has been extensively investigated as it plays a role in regulation of cellular respiration and pathophysiology. Cytochrome bd is a tri-heme (b₅₅₈, b₅₉₅, d) bacterial oxidase containing no copper that couples electron transfer from quinol to O₂ (to produce H₂O) with generation of a transmembrane protonmotive force. In this work, we investigated by stopped-flow absorption spectroscopy the reaction of NO with Escherichia coli cytochrome bd in the fully oxidized (O) state. We show that under anaerobic conditions, the O state of the enzyme binds NO at heme d with second-order rate constant k_on = 1.5 ± 0.2 × 10² M⁻¹ s⁻¹, yielding a nitrosyl adduct (d³⁺–NO or d²⁺–NO⁺) with characteristic optical features (an absorption increase at 639 nm and a red shift of the Soret band). The reaction mechanism is remarkably different from that of O cytochrome c oxidase in which the heme–copper binuclear center reacts with NO approximately three orders of magnitude faster, forming nitrite. The data allow us to conclude that in the reaction of NO with terminal oxidases in the O state, Cu_B is indispensable for rapid oxidation of NO into nitrite.     

Electron transfer after flash photolysis of mixed-valence carboxycytochrome c oxidase

The light-induced difference spectra of the fully reduced (a³⁺a²⁺₃-CO) complex and the mixed-valence carboxycytochrome c oxidase (a³⁺a²⁺₃-CO) during steady-state illumination and after flash photolysis showed marked differences. The differences appear to be due to electron transfer between the redox centres in the enzyme. The product of the absorbance coefficient and the quantum yield was found to be equal in both enzyme species, both when determined from the rates of photolysis and from the values of the dissociation constants of the cytochrome a²⁺₃-CO complex. This would confirm that the spectral properties of cytochrome a₃ are not affected by the redox state of cytochrome a and Cu_A. When the absorbance changes after photolysis of cytochrome a²⁺₃-CO with a laser flash were followed on a time scale from 1 μs to 1 s in the fully reduced carboxycytochrome c oxidase, only the CO recombination reaction was observed. However, in the mixed-valence enzyme an additional fast absorbance change (k = 7·10³s^?1) was detected. The kinetic difference spectrum of this fast change showed a peak at 415 nm and a trough at 445 nm, corresponding to oxidation of cytochrome a₃. Concomitantly, a decrease of the 830 nm band was observed due to reduction of Cu_A. This demonstrates that in the partially reduced enzyme a pathway is present between Cu_A and the cytochrome a₃-Cu_B pair, via which electrons are transferred rapidly.     

EPR studies of the photodissociation reactions of cytochrome c oxidase-nitric oxide complexes

Three complexes of NO with cytochrome c oxidase are described which are all photodissociable at low temperatures as measured by EPR. The EPR parameters of the cytochrome a²⁺₃-NO complex are the same both in the fully reduced enzyme and in the mixed-valence enzyme. The kinetics of photodissociation of cytochrome a²⁺₃-NO and recombination of NO with cytochrome a²⁺₃ (in the 30–70 K region) revealed no differences in structure between cytochrome a²⁺₃ in the fully reduced and the mixed-valence states. The action spectrum of the photodissociation of cytochrome a²⁺₃-NO as measured by EPR has maxima at 595, 560 and 430 nm, and corresponds to the absorbance spectrum of cytochrome a²⁺₃-NO. Photodissociation of cytochrome a²⁺₃-NO in the mixed-valence enzyme changes the EPR intensity at g 3.03, due to electron transfer from cytochrome a²⁺₃ to cytochrome a³⁺. The extent of electron transfer was found to be temperature dependent. This suggests that a conformational change is coupled to this electron transfer. The complex of NO with oxidized cytochrome c oxidase shows a photodissociation reaction and recombination of NO (in the 20–40 K region) which differ completely from those observed in cytochrome a²⁺₃-NO. The observed recombination occurs at a temperature 15 K lower than that found for the cytochrome a²⁺₃-NO complex. The action spectrum of the oxidized complex shows a novel spectrum with maxima at 640 and below 400 nm; it is assigned to a Cu²⁺_B-NO compound. The triplet species with Δm_s = 2 EPR signals at g 4 and Δm_s = 1 signals at g 2.69 and 1.67, that is observed in partially reduced cytochrome c oxidase treated with azide and NO, can also be photodissociated.     

Peculiarities of cyanide binding to the <Emphasis Type="Italic">ba</Emphasis><Subscript>3</Subscript>-type cytochrome oxidase from the thermophilic bacterium <Emphasis Type="Italic">Thermus thermophilus</Emphasis>

Cytochrome c oxidase of the ba ₃-type from Thermus thermophilus does not interact with cyanide in the oxidized state and acquires the ability to bind heme iron ligands only upon reduction. Cyanide complexes of the reduced heme a ₃ in cytochrome ba ₃ and in mitochondrial aa ₃-type cytochrome oxidase are similar spectroscopically, but the a ₃²⁺-CN complex of cytochrome ba ₃ is strikingly tight. Experiments have shown that the K _d value of the cytochrome ba ₃ complex with cyanide in the presence of reductants of the enzyme binuclear center does not exceed 10⁻⁸ M, which is four to five orders of magnitude less than the K _d of the cyanide complex of the reduced heme a ₃ of mitochondrial cytochrome oxidase. The tightness of the cytochrome ba ₃ complex with cyanide is mainly associated with an extremely slow rate of the ligand dissociation (k _off ≤ 10⁻⁷ sec⁻¹), while the rate of binding (k _on ∼ 10² M⁻¹·sec⁻¹) is similar to the rate observed for the mitochondrial cytochrome oxidase. It is proposed that cyanide dissociation from the cytochrome ba ₃ binuclear center might be hindered sterically by the presence of the second ligand molecule in the coordination sphere of Cu_B²⁺. The rate of cyanide binding with the reduced heme a ₃ does not depend on pH in the neutral area, but it approaches linear dependence on H⁺ activity in the alkaline region. Cyanide binding appears to be controlled by protonation of an enzyme group with pK _a = 8.75.     

Synthetic and mechanistic studies on ruthenium dicarbonyl complexes containing PhP(CH2CH2CH2PCy2)2 (Cyttp) ligand

A synthetic and mechanistic study is reported on ligand substitution and other reactions of six-coordinate ruthenium(II) carbonyl complexes containing tridentate PhP(CH₂CH₂CH₂PCy₂)₂ (Cyttp). Carbonylation of cis-mer-Ru(OSO₂CF₃)₂(CO)(Cyttp) (1) affords [cis-mer-Ru(OSO₂CF₃)(CO)₂(Cyttp)]O₃SCF₃ (2(O₃SCF₃)) and, on longer reaction times, [cis-mer-Ru(solvent)(CO)₂(Cyttp)](O₃SCF₃)₂ (solvent = acetone, THF, methanol). 2(O₃SCF₃) reacts with each of NaF, LiCl, LiBr, NaI, and LiHBEt₃ to yield [cis-mer-RuX(CO)₂(Cyttp)]⁺ (X = F (3), Cl (4), Br (5), I (6), H (7)), isolated as 3-7(BPh₄). These conversions proceed with high stereospecificity to afford only a single isomer of the product that is assigned a structure in which the Ph group of Cyttp points toward the CO trans to X (anti when X = F, Cl, Br, or I; syn when X = H). Treatment of 2(O₃SCF₃) with NaOMe and CO generates the methoxycarbonyl complex [cis-mer-Ru(CO₂Me)(CO)₂(Cyttp)]⁺ (8), whereas addition of excess n-BuLi to 2(O₃SCF₃) in THF under CO affords mer-Ru(CO)₂(Cyttp) (9). The two ¹³C isotopomers [cis-mer-Ru(OSO₂CF₃)(CO)(¹³CO)(Cyttp)]O₃SCF₃ (2′(O₃SCF₃): ¹³CO trans to P_C; 2″(O₃SCF₃): ¹³CO cis to all P donors) were synthesized by appropriate adaptations of known transformations and used in mechanistic studies of reactions with each of LiHBEt₃, NaOMe/CO, and n-BuLi. Whereas LiHBEt₃ reacts with 2′(O₃SCF₃) and 2″(O₃SCF₃) to replace triflate by hydride without any scrambling of the carbonyl ligands, the corresponding reactions of NaOMe-CO are more complex. The methoxide combines with the CO cis to triflate in 2, and the resultant methoxycarbonyl ligand ends up positioned trans to the incoming CO in 8. A mechanism is proposed for this transformation. Finally, treatment of either 2′(O₃SCF₃) or 2″(O₃SCF₃) with an excess of n-BuLi leads to the formation of the same two ruthenium(0) isomers of mer-Ru(CO)(¹³CO)(Cyttp). These products represent, to our knowledge, the first example of a syn-anti pair of isomers of a five-coordinate metal complex.     

Structure and coordination of CuB in the Acidianus ambivalens aa 3 quinol oxidase heme–copper center

The coordination environment of the Cu_B center of the quinol oxidase from Acidianus ambivalens, a type B heme–copper oxygen reductase, was investigated by Fourier transform (FT) IR and extended X-ray absorption fine structure (EXAFS) spectroscopy. The comparative structural chemistry of dinuclear Fe–Cu sites of the different types of oxygen reductases is of great interest. Fully reduced A. ambivalens quinol oxidase binds CO at the heme a ₃ center, with ν(CO)=1,973 cm⁻¹. On photolysis, the CO migrated to the Cu_B center, forming a Cu_B^I–CO complex with ν(CO)=2,047 cm⁻¹. Raising the temperature of the samples to 25°C did not result in a total loss of signal in the FTIR difference spectrum although the intensity of these signals was reduced sevenfold. This observation is consistent with a large energy barrier against the geminate rebinding of CO to the heme iron from Cu_B, a restricted limited access at the active-site pocket for a second binding, and a kinetically stable Cu_B–CO complex in A. ambivalens aa ₃. The Cu_B center was probed in a number of different states using EXAFS spectroscopy. The oxidized state was best simulated by three histidines and a solvent O scatterer. On reduction, the site became three-coordinate, but in contrast to the bo ₃ enzyme, there was no evidence for heterogeneity of binding of the coordinated histidines. The Cu_B centers in both the oxidized and the reduced enzymes also appeared to contain substoichiometric amounts (0.2 mol equiv) of nonlabile chloride ion. EXAFS data of the reduced carbonylated enzyme showed no difference between dark and photolyzed forms. The spectra could be well fit by 2.5 imidazoles, 0.5 Cl⁻ and 0.5 CO ligands. This arrangement of scatterers would be consistent with about half the sites remaining as unligated Cu(his)₃ and half being converted to Cu(his)₂Cl⁻CO, a 50/50 ratio of Cu(his)₂Cl⁻ and Cu(his)₃CO, or some combination of these formulations. Electronic Supplementary Material Supplementary material is available for this article at .     

Electron and proton transfer in the <Emphasis Type="Italic">ba</Emphasis><Subscript>3</Subscript> oxidase from <Emphasis Type="Italic">Thermus thermophilus</Emphasis>

The ba ₃-type cytochrome c oxidase from Thermus thermophilus is phylogenetically very distant from the aa ₃–type cytochrome c oxidases. Nevertheless, both types of oxidases have the same number of redox-active metal sites and the reduction of O₂ to water is catalysed at a haem a ₃-Cu_B catalytic site. The three-dimensional structure of the ba ₃ oxidase reveals three possible proton-conducting pathways showing very low homology compared to those of the mitochondrial, Rhodobacter sphaeroides and Paracoccus denitrificans aa ₃ oxidases. In this study we investigated the oxidative part of the catalytic cycle of the ba ₃ -cytochrome c oxidase using the flow-flash method. After flash-induced dissociation of CO from the fully reduced enzyme in the presence of oxygen we observed rapid oxidation of cytochrome b (k ≅ 6.8 × 10⁴ s⁻¹) and formation of the peroxy (P_R) intermediate. In the next step a proton was taken up from solution with a rate constant of ~1.7 × 10⁴ s⁻¹, associated with formation of the ferryl (F) intermediate, simultaneous with transient reduction of haem b. Finally, the enzyme was oxidized with a rate constant of ~1,100 s⁻¹, accompanied by additional proton uptake. The total proton uptake stoichiometry in the oxidative part of the catalytic cycle was ~1.5 protons per enzyme molecule. The results support the earlier proposal that the P_R and F intermediate spectra are similar (Siletsky et al. Biochim Biophys Acta 1767:138, 2007) and show that even though the architecture of the proton-conducting pathways is different in the ba ₃ oxidases, the proton-uptake reactions occur over the same time scales as in the aa ₃-type oxidases. Smirnova and Zaslavsky contributed equally to the work described in this paper.     

6-Halogenopyridin-2-olato complexes with the diruthenium(2+) core: Equilibria between head-to-head and head-to-tail structures

The dinuclear bis(6-X-pyridin-2-olato) ruthenium complexes [Ru₂(μ-XpyO)₂(CO)₄(PPh₃)₂] (X = Cl (4B) and Br (5B)), [Ru₂(μ-XpyO)₂(CO)₄(CH₃CN)₂] (X = Cl (6B), Br (7B) and F (8B)) and [Ru₂(μ-ClpyO)₂(CO)₄(PhCN)₂] (9B) were prepared from the corresponding tetranuclear coordination dimers [Ru₂(μ-XpyO)₂(CO)₄]₂ (1: X = Cl; 2: X = Br) and [Ru₂(μ-FpyO)₂(CO)₆]₂ (3) by treatment with an excess of triphenylphosphane, acetonitrile and benzonitrile, respectively. In the solid state, complexes 4B-9B all have a head-to-tail arrangement of the two pyridonate ligands, as evidenced by X-ray crystal structure analyses of 4B, 6B and 9B, in contrast to the head-to-head arrangement in the precursors 1-3. A temperature- and solvent-dependent equilibrium between the yellow head-to-tail complexes and the red head-to-head complexes 4A-7A and 9A, bearing an axial ligand only at the O,O-substituted ruthenium atom, exists in solution and was studied by NMR spectroscopy. Full ¹H and ¹³C NMR assignments were made in each case. Treatment of 1 and 2 with the N-heterocyclic carbene (NHC) 1-butyl-3-methylimidazolin-2-ylidene provided the complexes [Ru₂(μ-XpyO)₂(CO)₄(NHC)], X = Cl (11A) or Br (12A). An XRD analysis revealed the head-to-head arrangement of the pyridonate ligands and axial coordination of the carbene ligand at the O,O-substituted ruthenium atom. The conversion of 11A and 12A into the corresponding head-to-tail complexes was not possible.     

Ligand Trapping by Cytochrome c Oxidase: IMPLICATIONS FOR GATING AT THE CATALYTIC CENTER*

Cytochrome c oxidase is a member of the heme-copper family of oxygen reductases in which electron transfer is linked to the pumping of protons across the membrane. Neither the redox center(s) associated with proton pumping nor the pumping mechanism presumably common to all heme-copper oxidases has been established. A possible conformational coupling between the catalytic center (Fe_a3³⁺–Cu_B²⁺) and a protein site has been identified earlier from ligand binding studies, whereas a structural change initiated by azide binding to the protein has been proposed to facilitate the access of cyanide to the catalytic center of the oxidized bovine enzyme. Here we show that cytochrome oxidase pretreated with a low concentration of azide exhibits a significant increase in the apparent rate of cyanide binding relative to that of free enzyme. However, this increase in rate does not reflect a conformational change enhancing the rapid formation of a Fe_a3³⁺–CN–Cu_B²⁺ complex. Instead the cyanide-induced transition of a preformed Fe_a3³⁺–N₃–Cu_B²⁺ to the ternary complex of Fe_a3³⁺–N₃ Cu_B²⁺–CN is the most likely reason for the observed acceleration. Significantly, the slow rate of azide release from the ternary complex indicates that cyanide ligated to Cu_B blocks a channel between the catalytic site and the solvent. The results suggest that there is a pathway that originates at Cu_B and that, during catalysis, ligands present at this copper center control access to the iron of heme a₃ from the bulk medium.     

Electron-transfer processes in carboxy-cytochrome c oxidase after photodissociation of cytochrome a2+3 · CO

Under continuous illumination the CO binding curve of reduced carboxy-cytochrome c oxidase maintains the shape of the binding curve in the dark. The apparent dissociation constant calculated from the binding curves at various light intensities is a linear function of the light intensity.Marked differences are observed between the light-induced difference spectra of the fully reduced carboxy-cytochrome c oxidase and the mixed-valence carboxy-cytochrome c oxidase. These differences are enhanced in the presence of ferricyanide as an electron acceptor and are explained by partial oxidation of cytochrome a₃ in the mixed-valence enzyme after photodissociation.Upon addition of CO to partially reduced formate cytochrome c oxidase (a²⁺a³⁺₃ · HCOOH) the cytochrome a²⁺₃ · CO compound is formed completely with a concomitant oxidation of cytochrome a and the Cu associated with cytochrome a. During photodissociation of the CO compound the formate rebinds to cytochrome a₃ and cytochrome a and its associated Cu are simultaneously reduced. These electron transfer processes are fully reversible since in the dark the a³⁺₃ · HCOOH compound is dissociated slowly with a concomitant formation of the a²⁺₃ · CO compound and oxidation of cytochrome a.When these experiments are carried out in the presence of cytochrome c, both cytochrome c and cytochrome a are reduced upon illumination of the mixed-valence carboxy-cytochrome c oxidase. In the dark both cytochrome c and cytochrome a are reoxidized when formate dissociates from cytochrome a₃ and the a²⁺₃ · CO compound is formed back. Thus, in this system we are able to reverse and to modulate the redox state of the different components of the final part of the respiratory chain by light.     

An optical material for the detection of trace S<Subscript>2</Subscript>O<Subscript>3</Subscript><Superscript>2−</Superscript> in milk based on a copper complex

A novel S₂O₃ ^2? luminescent sensor (Cu²⁺-p-CPIP) was developed and the presence of S₂O₃ ^2? caused an obvious fluorescence enhancement at 420 nm upon excitation at 330 nm, which could be distinguished with the naked eye under a UV lamp. Remarkably, the compound exhibited excellent selective and sensitive response to S₂O₃ ^2? over other common anions with a micromolar limit of detection (0.442 μM) in DMSO/H₂O (v/v, 1:1) buffer. The absorbance intensity and the color of Cu²⁺-p -CPIP solution changed gradually with the increase of S₂O₃ ^2? concentration. The proposed method was applied to the determination of S₂O₃ ^2? in milk samples and the recoveries were 97.5–105%. The preparation of Cu²⁺-p -CPIP exhibited the quick, simple and facile advantages. The results showed that Cu²⁺-p -CPIP can be a good candidate for simple, rapid and sensitive colorimetric detection of S₂O₃ ^2? in aqueous solution.     

Syntheses, crystal structures and magnetic properties of trinuclear and [3 × 1 + 1 × 2] pentanuclear complexes derived from a compartmental ligand: Role of solvent on nuclearity and number of components

Reaction of [Cu^IIL⊂(H₂O)] (H₂L = N,N′-ethylenebis(3-ethoxysalicylaldimine)) with nickel(II) perchlorate in 1:1 ratio in acetone produces the trinuclear compound [(Cu^IIL)₂Ni^II(H₂O)₂](ClO₄)₂ (1). On the other hand, on changing the solvent from acetone to methanol, reaction of the same reactants in same ratio produces the pentametallic compound [(Cu^IIL)₂Ni^II(H₂O)₂](ClO₄)₂·2[Cu^IIL⊂(H₂O)]·2MeOH (2A), which loses solvated methanol molecules immediately after its isolation to form [(Cu^IIL)₂Ni^II(H₂O)₂](ClO₄)₂·2[Cu^IIL⊂(H₂O)] (2B). Clearly, formation of 1 versus 2A and 2B is solvent dependent. Crystal structures of 1 and 2A have been determined. Interestingly, compound 2A is a [3 × 1 + 1 × 2] cocrystal. The cryomagnetic profiles of 1 and 2B indicate that the two pairs of copper(II)···nickel(II) ions in the trinuclear cores in both the complexes are coupled by almost identical moderate antiferromagnetic interaction (J = −22.8 cm⁻¹ for 1 and −26.0 cm⁻¹ for 2B).