Recombination Of Carbon Monoxide Research Articles

Photoinduced transport in an H64Q neuroglobin antidote for carbon monoxide poisoning.

Carbon monoxide (CO) is a leading cause of poisoning deaths worldwide, without available antidotal therapy. Recently, a potential treatment for CO poisoning was introduced, based on binding of CO by neuroglobin (Ngb) with a mutated distal histidine (H64Q). Here, we present an atomistic mechanism of CO trapping by H64Q Ngb revealed by nonadiabatic molecular dynamics. We focused on CO photodissociation and recombination of CO to wild type (WT) and H64Q Ngb. Our results demonstrate that the distribution of CO within the proteins differs substantially due to rearrangement of amino acids surrounding the distal heme pocket. This leads to the decrease of the distal pocket volume in H64Q Ngb in comparison to WT Ngb, trapping migrating CO molecules in the distal pocket. We show that the mutation implicates the shortening of the time scale of CO geminate recombination, making H64Q Ngb 2.7 times more frequent binder than WT Ngb.

Low pressure glow-discharge methanation with an ancillary oxygen ion conductor

Plasma technology is gaining increased interest for the reduction of carbon dioxide (CO2) to carbon monoxide (CO) and oxygen (O2). CO is a reaction partner with hydrogen (H2) to produce gas and fuel. This paper elaborates an improved method to produce methane (CH4) by plasma induced dissociation of carbon dioxide and H2 in one single setup. A low pressure glow discharge plasma with an integrated zirconia based ion conductor transports oxygen ions (O2−) in or out of the reaction chamber. Transporting oxygen ions into the reaction chamber is a way to have a controlled amount of water vapor for the conducted experiments. In contrast, leads the reduced amount of O2 ions to a theoretical decrease in the recombination of CO and O2 to CO2 in the ionized gas and therefore increases the CO2 conversion rate. The applied voltage to the ion conductor for the outward ion transportation and the power of the plasma source current have a major impact on the amount of extracted O2− ions. The effect of these parameters on the CO production rate and the formation of methane are investigated. A positive effect of the plasma current on the amount of CO and CH4 is detected. The influence of the temperature increase from the heated ion conductor relates to an undesired increase of the recombination rate of CO and O2 to CO2, but has a positive effect on the methanation.

CO Photodissociation Dynamics in Cytochrome P450BM3 Studied by Subpicosecond Visible and Mid-Infrared Spectroscopy

Cytochrome P450BM3 is a bacterial enzyme with a heme cofactor that binds small diatomic ligands. Here we report the first study of carbon monoxide (CO) photodissociation and rebinding in ferrous P450BM3 on an ultrafast time scale. We monitored dissociation of carbon monoxide upon Soret band excitation using visible and infrared femtosecond spectroscopy between 100 fs and 4 ns. The dynamics of the ferric P450 was probed for reference in the visible spectral region. In the photodissociated ferrous P450-CO complex, the vibrational hot deligated ground state is populated in 0.2 ps and relaxes on a picosecond time scale. The onset of geminate recombination of CO with the heme is observed on a nanosecond time scale. In the mid-infrared spectral region, the bleached absorption due to the bound C=O stretch vibration is constant on the picosecond to 1 ns time scale, indicating that the photodissociation yield is 100% and that rebinding occurs after 1 ns. In the infrared absorption difference spectra, we additionally resolve two small bands of dissociated CO molecules at 2092 and 2114 cm(-1). This indicates that the escape of photolyzed CO to solvent and the geminate recombination are preceded by transient docking within the protein in a manner similar to that of globins. The bands partially decay with a time constant of 1 ps, possibly due to a relaxation of the protein around the CO docking site, allowing for greater orientational freedom of the CO molecules.

Conformational Dependence of Hemoglobin Reactivity under High Viscosity Conditions: The Role of Solvent Slaved Dynamics

The concept of protein dynamic states is introduced. This concept is based on (i) protein dynamics being organized hierarchically with respect to solvent slaving and (ii) which tier of dynamics is operative over the time window of a given measurement. The protein dynamic state concept is used to analyze the kinetic phases derived from the recombination of carbon monoxide to sol-gel-encapsulated human adult hemoglobin (HbA) and select recombinant mutants. The temperature-dependent measurements are made under very high viscosity conditions obtained by bathing the samples in an excess of glycerol. The results are consistent with a given tier of solvent slaved dynamics becoming operative at a time delay (with respect to the onset of the measurement) that is primarily solvent- and temperature-dependent. However, the functional consequences of the dynamics are protein- and conformation-specific. The kinetic traces from both equilibrium populations and trapped allosteric intermediates show a consistent progression that exposes the role of both conformation and hydration in the control of reactivity. Iron-zinc symmetric hybrid forms of HbA are used to show the dramatic difference between the kinetic patterns for T state alpha and beta subunits. The overall results support a model for allostery in HbA in which the ligand-binding-induced transition from the deoxy T state to the high -affinity R state proceeds through a progression of T state intermediates.

Structural dynamics of myoglobin: an infrared kinetic study of ligand migration in mutants YQR and YQRF

Recombination of carbon monoxide to myoglobin mutants YQR and YQRF was studied using transient infrared absorption spectroscopy and Fourier transform infrared–temperature derivative spectroscopy (FTIR–TDS). Photoproduct states B, C′, C″ and D associated with ligands residing in different protein cavities have been identified. After photolysis, ligands migrate to primary docking site B and subsequently rebind or escape to a secondary site (C) within the Xe4 cavity. For YQR, a global analysis of the isothermal rebinding kinetics below 160 K and the TDS data reveal a correlation between the enthalpy barriers governing the two processes. Above 120 K, a protein conformational change in both YQR and YQRF converts photoproduct C′ into C″ with markedly slowed kinetics. Above ∼180 K, ligands migrate to the proximal Xe1 site (D) and also exit into the solvent, from where they rebind in a bimolecular reaction.

Modelling spin-forbidden reactions: recombination of carbon monoxide with iron tetracarbonyl.

New density functional theory and ab initio computations on the [Fe(CO)5] system are reported. Careful exploration of basis set and correlation effects leads to "best" values for the difference in energy deltaE(1,3) between ground state 3[Fe(CO)4] and the singlet excited state of ca. 8 kcal mol(-1), and for the bond dissociation energy BDE(3) of [Fe(CO)5] with respect to ground state fragments 3[Fe(CO)4] + CO of ca. 40 kcal mol(-1). A modified form of the B3PW91 functional is used to explore the potential energy surface for the spin-forbidden recombination reaction of CO with 3[Fe(CO)4]. A Cs-symmetric minimum energy crossing point (MECP) between the reactant (triplet) and product (singlet) potential energy surfaces is found, lying 0.43 kcal mol(-1) above the reactants. The rate coefficient for recombination is computed using a non-adiabatic form of transition state theory, in which the MECP is treated as the critical point in the reaction. Semi-quantitative agreement with experiment is obtained: the predicted rate coefficient, 8.8 x 10(-15) cm3 molecule(-1) s(-1), is only six times smaller than the experimental rate. This is the first computation from first principles of a rate coefficient for a spin-forbidden reaction of a transition metal compound.

Behaviour of catalytic probe during surface activation of polyether sulphone

The influence of a polymer sample on the behaviour of a catalytic probe was determined. Highly dissociated oxygen plasma was created in a glass cylindrical tube with an inductively coupled RF generator. The density of oxygen atoms was determined with a nickel catalytic probe at different pressures between 20 and 400 Pa. A polymer sample with different area was placed in the discharge vessel and the signal of the catalytic probe was measured at different pressure. During the treatment of polymer samples in oxygen plasma it was found that the catalytic probe signal depended on the area of the samples exposed to plasma. The probe signal was higher in the presence of polymer than in the case of an empty discharge vessel. The signal of the catalytic probe increased monotonically with an increasing surface area of the polymer. The increase of the probe signal was explained by additional heating of the probe due to recombination of carbon monoxide on its surface.

Laser photolysis behavior of ferrous horseradish peroxidase with carbon monoxide and cyanide: effects of mutations in the distal heme pocket.

Native horseradish peroxidase and several forms with mutations in the distal heme pocket (His42Leu, His42Arg, and Arg38Leu) have been expressed in Escherichia coli. These enzymes have been purified and analyzed in terms of the room temperature recombination rate of carbon monoxide and cyanide after photolysis of the reduced forms. The recombinant wild-type ferrous form exhibited monophasic recombination of carbon monoxide with an observed bimolecular rate constant at pH 8.5 of 4.4 x 10(3) M-1 s-1 which is essentially the same as the natural glycosylated form. This recombination rate constant increases in the mutants in the order WT < H42R < H42L << R38L. The value for R38L (5 x 10(6) M-1 s-1) is increased by 3 orders of magnitude relative to the wild-type and is similar to that for human hemoglobin [Mims et al. (1983) J. Biol. Chem. 258, 14219-14232]. Cyanide recombination with the wild-type ferrous form at room temperature is biphasic at pH 6.5 but becomes more monophasic at pH 8.5, again similar to the behavior of the natural glycosylated form, although the Fe(2+)-cyano form of the recombinant enzyme appears to be more unstable at high pH. None of the mutant forms were able to bind cyanide in the ferrous state to any significant extent (Kdiss > 250 mM) when cyanide was added at a concentration (10-20 mM) sufficient to almost saturate the wild-type form (Kdiss approximately equal to 1 mM at pH 7). This behavior contrasts with that of the oxidized forms of the mutants where increases in cyanide dissociation constants are smaller ( < 25 times).(ABSTRACT TRUNCATED AT 250 WORDS)

The binding of cyanide and carbon monoxide to bacterial cytochrome bo

Introduction Cytochrome (cyt) bo from Exherichia coli is a member of a superfamily of homologous protonmotive oxidases which includes mammalian cyt c oxidase [ l ] . It has become evident that the copperhaem binuclear centre is very similar in all of these oxidases even though the haem type and reducing substrate vary. This binuclear centre is the site of oxygen binding and reduction to water, and the notion that local ligand movements in the binuclear centre drive proton translocation between proton channels in the protein structure [21 has become widely adopted as the most likely working model for the primary chemistry that drives vectorial proton translocation (for detailed variants on this theme, see [ 3-71). We have embarked on a detailed study of ligand binding dynamics in order to gain further insight into such possible protonmotive chemistry. Site-directed mutagenesis in bacterial systems o f conserved possible metal ligands allowed fairly definitive assignment of four histidine ligands to the three metal centres (two haem groups and one copper, Cu,,) in the critical subunit I of these oxidases [ X,9 1. Further biophysical analyses led to reassignment of the two remaining conserved histidines of subunit 1, together with the identification of other amino acids which might influence the metal centre chemistry. A summary of the view reached by our own laboratory of the possible ligands around Cu,,. based on data in [X,O] and on our measurements of mutation effects on recombination o f carbon monoxide after laser photolysis [lo], is shown in Figure 1. Complementary types of spectroscopic data from other laboratories have produced a remarkably consistent model [ 11-13]. We found that mutation of two histidine ligands (H333I,, H3341,) led to an acceleration of the rate of recombination of carbon monoxide with the binuclear centre [lo], a result also observed if the wild-type enzyme is grown in copper-limited conditions so that Cull is absent [14,15]. We have deduced that an acceleration of recombination rate of fivefold or more can be taken as a strong indication that Cu,, is absent. Indeed, we have often observed heterogeneity of CO recombination in

Microscopic derivation of the low‐T myoglobin‐CO recombination rate law by estimating statistical parameters of folding relaxation

AbstractThe low‐T recombination of carbon monoxide (CO) to myoglobin (Mb) obeys a rate law interpreted as a superposition of appropriately‐weighted Arrhenius‐type relaxations. We derive this rate law from microscopic principles by determining the random activation energy landscape for short timescale interconversion of myoglobin conformational substates that preserve CO‐storage capacity. Thus, the random energy model (REM) first introduced in the context of protein folding by Bryngelson and Wolynes is applied here to derive the nonexponential rate law of a reaction ‐ Mb‐CO recombination — built upon an REM‐type distribution of substate reactants.

Flash photolysis of the carbon monoxide compounds of wild-type and mutant variants of cytochrome bo from Escherichia coli

The carbon monoxide compounds of the fully reduced and mixed valence forms of cytochrome bo from Escherichia coli were laser photolysed under anaerobic conditions at room temperature. The carbon monoxide recombined with characteristic rate constants of 50 s −1 or 35 s −1 in the fully reduced and mixed valence forms, respectively. Rates of CO recombination with the fully reduced enzyme were examined in a variety of mutant forms of cytochrome bo, produced by site-directed mutagenesis. A method was developed to deconvolute cytochromes bo and bd, leading to some reassessment of histidine ligands to the metals. Significant changes in the rate constant of recombination of carbon monoxide occurred in many of these mutants and these results could be rationalised generally in terms of our current working model of the folding structure of subunit I. In the mixed valence form of the enzyme the transient photolysis spectra in the visible region are consistent with a rapid electron redistribution from the binuclear centre to the low-spin haem. This electron transfer is biphasic, with rate constants of around 10 5 and 800 s −1. The process was also examined in the His-33-Leu mutant, in which a putative histidine ligand to Cu B is replaced by leucine, and which results in the loss of the Cu B. It appeared that rapid haem-haem electron transfer could still occur. The observation that Cu B is apparently not required for rapid haem-haem electron transfer is consistent with the recently proposed model in which the two haems are positioned on opposite sides of transmembrane helix X in subunit I of the oxidase.

Rapid screening of cytochromes of respiratory mutants of Saccharomyces cerevisiae

A technique has been developed for the direct analysis by visible spectrophotometry of yeast spots growing on agar plates. This allows rapid semi-quantitative estimations of cytochromes c, b and oxidase and permits the identification of strains with impaired respiratory electron flow. Results of screening of 105 mutants are presented. There appears to be a correlation between the exonic location of the mutation in COX1 of oxidase and the level of optically detectable enzyme. Mutations in cytochrome b of the bc1 complex also affect the level of expression of cytochrome oxidase and can cause either an increased or decreased level of expression of oxidase relative to the wild-type strain. Twelve strains selected by the rapid level-1 screening were grown as lawns on sections of an agar plate and resuspended for a second level of screening. Quantitative estimates have been made of the concentrations of cytochromes, the turnover number of cytochrome oxidase and the kinetics of recombination of carbon monoxide with oxidase after flash photolysis. This confirmed the validity of the rapid screening procedure, and we have identified several strains which contain high levels of a mutant form of cytochrome oxidase with properties worthy of further investigation.

Molecular tunneling and pumping effects in low temperature MBCO recombination

Recombination of carbonmonoxide after photodissociation has been studied by Mossbauer spectroscopy at 4.2K and in the low temperature region, where tunneling effects play an important role in rebinding. We interpret the kinetic results in terms of a radiationless nonadiabatic multiphonon transition, which leads to a uniform description for all temperatures. Prolonged illumination at low temperature results in pumping into long-living states.

Oxygen and carbon monoxide reactions with heme proteins: quantum yields and geminate recombination on picosecond time scales

Picosecond time-resolved absorption spectroscopy and low-temperature studies have been undertaken in order to understand the nature of the intrinsic quantum yields and geminate recombination of carbon monoxide and oxygen to hemoglobin and myoglobin. We find that the photoproduct yields at 40 ps and long times (minutes) after photolysis at 8 K are similar; however, the yield of oxygen photoproducts is 0.4 +/- 0.1 while the yield of carbon monoxide photoproducts is 1.0 +/- 0.1 for both myoglobin and hemoglobin. Measurements in the Soret, near-infrared, and far-IR are used to quantitate the photoproduct yields. These results call into question previous cryogenic kinetic studies of O2 recombination. Significant subnanosecond geminate recombination is observed in oxyhemoglobin down to 150 K, while below 100 K this geminate recombination disappears. The lower photoproduct yields for oxyheme protein complexes can be attributed to both subnanosecond and subpicosecond recombination events which are ligand and protein dynamics dependent.

Charge motion in MbCO crystals after flash photolysis

Charge motion accompanying the dissociation and recombination of carbon monoxide to oriented myoglobin crystals has been observed. The magnitude of the electrical signals detected after photodissociation by electrodes on either side of MbCO crystals of type A is consistent with the x-ray data showing that the doubly charged iron ion lies in the mean heme plane when a ligand is bound and moves out of the plane when deligated. Beyond 10 ms, the time development of the electrical signal is consistent with the kinetics observed optically after flash photolysis on the same crystals.

Enhancement of ligand binding to myoglobin by far-infrared excitation from a free-electron laser

We summarize the theoretical reasons for believing that far-infrared radiation in the 5–100-cm−1 range can influence ligand recombination in heme proteins. We explicitly use the nonadiabatic model to discuss both electronic and vibrational matrix elements. We present the results of our experiment carried on at the University of California, Santa Barbara, free-electron laser in which we observed a modification of the recombination of carbon monoxide with myoglobin at low temperatures due to 50.5-cm−1 irradiation.

Direct observation of the internal excitation in the products of the two-body recombination of carbon monoxide and tungsten pentacarbonyl

A transient infrared spectrum observed following the photolysis of gas phase W(CO) 6 in the presence of CO is shown to be due to internally excited W(CO) 6* formed by the two-body recombination of W(CO) 5 and CO. The internal excitation is shown to be predominantly vibrational.

Recombination of carbon monoxide to ferrous horseradish peroxidase types A and C

The recombination of carbon monoxide to isoenzymes A2 and C of horseradish peroxidase (HRP) was studied as a function of temperature (2 to 320 K) and pH (5 to 8.3) with flash photolysis and infrared difference absorption. At low temperatures three geminate recombination processes are observed. One of these internal processes, denoted by I∗, is exponential in time with a rate coefficient that deviates strongly from an Arrhenius behavior below 100 K, implying phonon-assisted tunneling. The two other processes, denoted by I, are non-exponential in time and related to different carbonyl isomers, as shown by the infrared difference spectra. The existence of three internal processes indicates that HRP differs considerably from myoglobin where only one internal process, I, is seen. Moreover, the internal processes in HRP are faster than process I in myoglobin. At 300 K, only one recombination process from the solvent is observed and it is very slow ( λ s ≈ 1 s −1 at 1 atm CO (1 atm = 101,325 Pa)), much slower than the corresponding association process in myoglobin. Since process I is fast, but binding from the solvent is slow, the barrier at the heme cannot be responsible for the small association rate. The infrared absorption difference spectra of the amide I/II bands indicate that photolysis and recombination trigger a two-step structural change. The slow recombination rate at 300 K can thus be explained by the large Gibbs energy of the conformational transition that is necessary to let CO move into the heme pocket. The partition coefficient for the CO in the heme pocket and the solvent is extremely small, while bond formation with the heme iron occurs in less than 100 nanoseconds.

Time-Resolved X-ray Absorption Spectroscopy of Carbon Monoxide-Myoglobin Recombination After Laser Photolysis

Results are presented for the first time-resolved x-ray absorption measurements with a time resolution of 300 microseconds on a dynamically evolving chemical system. By synchronizing a neodymium: yttrium-aluminum-garnet pulsed laser with the bursts of x-rays emitted from the Cornell High Energy Synchrotron Source, it was possible to monitor at room temperature the recombination of carbon monoxide with myoglobin after laser photolysis. Changes in the pre-edge structure and in the position of the iron edge of this protein were detected as a function of time.

Equilibrium, kinetic and structural properties of hemoglobin Cranston, an elongated β chain variant

Hemoglobin Cranston has an elongated β subunit owing to a frame shift mutation. Oxygen equilibrium measurements of stripped Hb Cranston ‡ ‡ abbreviation used: Hb, hemoglobin; Hb A, normal human adult hemoglobin; Hb Cr, hemoglobin Cranston; bis Tris, 2,2-bis(hydroxymethyl)-2,2′,2″-nitroloethanol; Tris, tris(hydroxymethyl)aminomethane; IHP, inositol hexaphosphate. at 20 °C in the absence of phosphate revealed a high affinity ( P 50 = 0·2 mm Hg at pH 7), non-co-operative hemoglobin variant with markedly reduced Böhr effect ( −Δ log P 50 Δ pH 7–8 = 0·2 ). The addition of inositol hexaphosphate resulted in an overall decrease in oxygen affinity ( P 50 = 0·7 mm Hg at pH 7), as well as an increase in co-operativity and Böhr effect ( −Δ log P 50 Δ pH 7–8 = 0·2 ). Rapid mixing and flash photolysis experiments reflected the equilibrium results. Over a pH range from 6 to 9 in the absence of phosphate, the rate of combination of carbon monoxide with Hb Cranston measured by a stopped-flow technique and following full or partial flash photolysis was extremely rapid ( l′, l′ 4, of ∼ 6 × 10 6 m −1s −1). In rapid kinetic experiments the addition of inositol hexaphosphate lowered the value of l′ to ∼ 0·5 × 10 6 m −1s −1 only after prior incubation with the deoxygenated protein. Inositol hexaphosphate had no effect on the rate of recombination of carbon monoxide following either full or partial flash photolysis. Overall oxygen dissociation and oxygen dissociation with carbon monoxide replacement, were measured and found to be slow ( k, k 4∼ 11 s −1), consistent with a high affinity hemoglobin. Sedimentation equilibrium experiments revealed that Hb Cranston, at concentrations used in the functional studies, is somewhat less tetrameric than Hb A but nonetheless does not exist solely as a non-co-operative dimer. These kinetic and centrifugational findings in conjunction with X-ray diffraction evidence suggested that a high affinity tetramer of Hb Cranston exists which may equilibrate slowly with inositol hexaphosphate. Oxygen equilibrium measurements, ligand binding kinetics and X-ray diffraction studies on equivalent mixtures of Hb Cranston and Hb A revealed an interaction between these two hemoglobins in vitro that most probably exists in vivo. The presence of asymmetric hybrid molecules, α 2 β A β Cranston, in the difference Fourier maps indicated that the hydrophobic tail of Hb Cranston is accommodated in the central cavity of the hybrid molecule between the two β chains and is relatively protected from the water environment, thus aiding in the stability of Hb Cranston in the red cell.