Kinetics Of CO Binding Research Articles

Chloride acts as a novel negative heterotropic effector of hemoglobin Rothschild (beta 37 Trp-->Arg) in solution.

The effects of chloride ion concentration on the rate constants for association of carbon monoxide with human hemoglobin A and a synthetic form of the mutant hemoglobin Rothschild (beta 37 Trp-->Arg) have been investigated by stopped-flow techniques. Previous studies of the structure [Kavanaugh et al. (1992) Biochemistry 31, 4111] and functional properties [Rivetti et al. (1993) Biochemistry 32, 2888] of hemoglobin Rothschild crystallized in the T state have demonstrated that the mutant arginine residues create new chloride ion binding sites and that chloride ions act to lower the oxygen affinity of hemoglobin Rothschild in these crystals. The studies reported here demonstrate a parallel effect of chloride ions on the rate of CO association with deoxygenated hemoglobin Rothschild in solution. Although the kinetics of CO binding to this hemoglobin in solution exhibit a Bohr effect, the chloride effect is independent of pH. In addition, we find that other halide ions have similar effects on the rate constants for the association of CO with this hemoglobin variant.

Kinetics of CO binding to cytochromes P450 in the endoplasmic reticulum.

The kinetics of CO binding to cytochromes P450 in rat liver microsomes were examined using the flash photolysis technique. Modulation of the kinetics by P450 form-specific effectors such as anti-P450 monoclonal antibodies and substrates was used to elucidate the kinetic behavior of individual P450s within the endoplasmic reticulum. The problem of attributing a kinetic parameter to a single P450 in the presence of multiple microsomal P450s was overcome with a difference method that employs the difference of the kinetic profiles obtained in the presence and absence of a P450 effector. Applying this approach to study the conformation/dynamics of P450 2B1 in microsomes from phenobarbital-treated rats revealed that the substrate benzphetamine enhances while testosterone inhibits the rate of CO binding to this P450. Similar experiments with P450 1A1 in microsomes from 3-methylcholanthrene-treated rats showed that the substrate benzo[a]pyrene accelerates CO binding. These results show that the access channel between solvent and heme in the P450 interior can be altered in a substrate- and P450-dependent manner to either hinder or facilitate CO diffusion to the heme iron. These results also demonstrate that analytical difference methods may be employed to characterize the conformation of individual P450s in their native membrane environment in the endoplasmic reticulum.

Kinetics of CO binding to putative Na +-motive oxidases of the o-type from Bacillus FTU and of the d-type from Escherichia coli

The kinetics of CO reassociation with isolated Bacillus FTU o-type oxidase and with solubilized membranes of Escherichia coli (GO 102 strain) containing the d-type oxidase only, upon laser flash photolysis under reducing conditions, were studied. In both cases, kinetics are shown to be composed of three phases (τ 35–70 μs, 0.25–0.5 ms and 2–5 ms). The spectra of the flash-induced absorbance changes of the first kinetic components proved to be characteristic of CO- o- and CO- b 595 d-cytochrome complexes in Bac. FTU and E.Coli, respectively. The spectra of the second and the third components appeared to be nearly the same in Bac. FTU and E.Coli with peaks for the former at 436–437 and 590 nm and troughs at 419–420 and 569 nm; and for the latter with peaks at 436–437 and 558–560 nm and troughs at 419–420 and 575–578 nm. The similarity between the putative Na +-pumping Bac. FTU o- and E. Coli d-type oxidases and their difference from the H +-motive Bac. FTU caa 3 − and E. Coli o-type oxidases are discussed.

Kinetics of CO binding to H +-motive oxidases of the caa3-type from Bacillus FTU and of the o-type from Escherichia coli

The kinetics of CO rebinding with isolated Bacillus FTU caa 3-type oxidase and with solubilized Escherichia coli membranes (GO103 strain) containing the o-type oxidase as the main O 2reducing enzyme were studied under reducing conditions by laser flash photolysis of the CO-oxidase complexes. The spectra of the optical absorbance changes upon photolysis were characteristic of CO- caa 3 and CO- o-oxidase complexes in Bac. FTU and E.Coli, respectively. Small quantities of d-type oxidase in E.Coli GO103 membranes were detected. The kinetics of CO reassociation with reduced caa 3 and o-type oxidases were monophasic with τ 25–30 ms in both cases.

Protein dynamics in minimyoglobin: is the central core of myoglobin the conformational domain?

The kinetics of CO binding to the horse myoglobin fragment Mb-(32-139), the so-called "mini-Mb," were investigated by laser flash photolysis in 0.1 M phosphate buffer and in buffer with 75% (vol/vol) glycerol. The reaction displays complex time courses that can be approximated satisfactorily only with a sum of five exponentials. The features of the kinetic components and a comparison of the deoxy-minus-carbonyl difference spectra of mini-Mb and horse Mb obtained under equilibrium conditions, with the kinetic difference spectra resulting from the global analysis of the traces recorded between 400 and 450 nm, show that CO binding to mini-Mb is accompanied by large structural changes. In view of the fact that mini-Mb is an approximation of the Mb-(31-105) fragment encoded by the central exon of the Mb gene, this finding is particularly relevant. On the basis of our data and previous reports [De Sanctis, G., Falcioni, G., Giardina, B., Ascoli, F. & Brunori, M. (1988) J. Mol. Biol. 200, 725-733; De Sanctis, G., Falcioni, G., Grelloni, F., Desideri, A., Polizo, F., Giardina, B., Ascoli, F. & Brunori, M. (1992) J. Mol. Biol. 222, 637-643], we propose that the protein fragment encoded by the central exon of the Mb gene is the domain responsible for ligand-linked conformational transitions, while the two terminal fragments dampen the amplitude of the structural changes that accompany ligand binding, thus rendering the protein stable and kinetically more efficient in its physiological function.

Expression of Pseudomonas aeruginosa nitrite reductase in Pseudomonas putida and characterization of the recombinant protein.

Nitrite reductase from Pseudomonas aeruginosa has been successfully expressed in Pseudomonas putida. The purified recombinant enzyme contains haem c but no haem d1. Nonetheless, like the holoenzyme from Ps. aeruginosa, it is a stable dimer (molecular mass 120 kDa), and electron transfer to oxidized azurin is biphasic and follows bimolecular kinetics (k1 = 1.5 x 10(5) and k2 = 2.2 x 10(4) M-1.s-1). Unlike the chemically produced apoenzyme, recombinant nitrite reductase containing only haem c is water-soluble, stable at neutral pH and can be quantitatively reconstituted with haem d1, yielding a holoenzyme with the same properties as that expressed by Ps. aeruginosa (namely optical and c.d. spectra, molecular mass, cytochrome c551 oxidase activity and CO-binding kinetics).

Rapid preparation of native alpha and beta chains of human hemoglobin

1. 1. Current procedures for the isolation of native chains of hemoglobin employ two ion exchange columns for each chain and result in readily autoxidizable chains with measurable contamination by Hb and Hg. 2. 2. In the new procedure, altered buffer conditions on the first column reduce Hb contamination from 2 to 5% to less than 1%, the limit of detectability. 3. 3. The second column and lengthy washes with beta mercaptoethanol are replaced by incubation with DTT for 1 min for alpha chains and, for beta chains, three incubations with DTT and separations by gel-filtration. The residual Hg is <0.1%. 4. 4. Oxidations in the previous procedure resulted in low yields and unreliable spectroscopic assessments of bound Hg. The new procedure resulted in a simple UV assay for Hg-free chains. 5. 5. Hemoglobin reconstituted from these oxy-chains was identical to native Hb in oxygen binding equilibria and in the kinetics of CO binding following laser photolysis.

Alteration of the proximal bond energy in the unliganded form of the homodimeric myoglobin from Nassa mutabilis Kinetic and spectroscopic evidence

CO binding kinetics to the homodimeric myoglobin (Mb) from Nassa mutabilis has been investigated between pH 1.9 and 7.O. Protonation of the proximal imidazole at low pH (≤ 3.0) and the consequent cleavage of the HisF8NE2-Fe proximal bond brings about a ▪ 20-fold increase of the second-order rate constant for CO binding. This process displays a p K a = 4.0 ± 0.2, significantly higher than that observed in all other deoxygenated hemoproteins investigated up to now. Such a feature underlies a decreased energy for the HisF8NE2-Fe proximal bond in the unliganded form and it also appears supported by resonance Raman spectroscopy in the low frequency region of the Fe(II) deoxygenated hemoprotein. Further, the pH-rate profile of N. mutabilis Mb, like that of the homodimeric hemoglobin (Hb) from Scapharca inaequivalvis (Coletta, M., Boffi, A., Ascenzi, P., Brunori, M. and Chiancone, E. (1990) J. Biol. Chem. 265, 4828–4830), can be described only by assuming a concerted proton-linked transition with n = 1.8 ± 0.1. Such a characteristic suggests, also on the basis of the amino acid sequence homology between N. mutabilis Mb and S. inaequivalvis Hb in the region forming the subunit interface, that the interaction mechanism is similar for the two homodimeric proteins, and drastically different from that operative in other hemoproteins.

The assignment of carbon monoxide association rate constants to the alpha and beta subunits in native and mutant human deoxyhemoglobin tetramers.

The association kinetics of CO binding to site-directed mutants of human deoxyhemoglobin were measured by stopped-flow rapid mixing techniques at pH 7.0, 20 degrees C. Hemoglobin tetramers were constructed from one set of native subunits and one set of mutated partners containing His(E7) to Gly, Val(E11) to Ala, or Val(E11) to Ile substitutions. The reactivity of beta Cys93 with p-hydroxymercuribenzoate was measured to ensure that the mutant deoxyhemoglobins were capable of forming T-state quaternary conformations. Time courses for the complete binding of CO were measured by mixing the deoxygenated proteins with a 5-fold excess of ligand in the absence and presence of inositol hexaphosphate. Association rate constants for the individual alpha and beta subunits in the T-state conformation were assigned by measuring time courses for the reaction of a small, limiting amount of CO with a 20-fold excess of deoxyhemoglobin (i.e. Hb4 + CO----Hb4(CO)). The effects of the E7 and E11 mutations in T-state alpha subunits were qualitatively similar to those observed for the same subunit in the R-state (Mathews, A.J., Rohlfs, R.J., Olson, J.S., Tame, J., Renaud, J-P., and Nagai, K. (1989) J. Biol. Chem. 264, 16573-16583). The alpha His58(E7) to Gly and Val62(E11) to Ala substitutions caused 80- and 3-fold increases, respectively, in k'CO for T-state alpha subunits, and the alpha Val62(E11) to Ile mutation caused a 3-fold decrease. The beta His63(E7) to Gly and Val67(E11) to Ala substitutions produced 70- and 8-fold increases, respectively, in k'CO for T-state beta subunits whereas these same mutations caused little effect on the rate of CO binding to R-state beta subunits. The beta Val67(E11) to Ile mutation produced the same large effect, a 23-fold reduction in k'CO, in both quaternary conformations of beta subunits. These kinetic results can be interpreted qualitatively in terms of differences between the alpha and beta subunits in the deoxy and liganded crystal structures of human hemoglobin (Perutz, M.F. (1990) Annu. Rev. Physiol. 52, 1-25). Both the structural and functional data suggest that the distal portion of the beta heme pocket is tightly packed in deoxyhemoglobin whereas the CO binding site in R-state beta subunits is much more open. In contrast, the distal portion of the alpha heme pocket is restricted sterically in both quaternary states.(ABSTRACT TRUNCATED AT 400 WORDS)

Oxidized dimeric Scapharca inaequivalvis. Co-driven perturbation of the redox equilibrium.

The dimeric hemoglobin isolated from Scapharca inaequivalvis, HbI, is notable for its highly cooperative oxygen binding and for the unusual proximity of its heme groups. We now report that the oxidized protein, an equilibrium mixture of a dimeric high spin aquomet form and a monomeric low spin hemichrome, binds ferrocyanide tightly which allows for internal electron transfer with the heme iron. Surprisingly, when ferricyanide-oxidized HbI is exposed to CO, its spectrum shifts to that of the ferrous CO derivative. Gasometric removal of CO leads to the oxidized species rather than to ferrous deoxy-HbI. At equilibrium, CO binds with an apparent affinity (p50) of about 10-25 mm of Hg and no cooperativity (20 degrees C, 10-50 mM buffers at pH 6.1). The kinetics of CO binding under pseudo-first order conditions are biphasic (t1/2 of 15-50 s at pH 6.1). The rates depend on protein, but not on CO concentration. The nitrite-oxidized protein is not reduced readily in the presence of CO unless one equivalent of ferrocyanide, but not of ferricyanide, is added. We infer that ferrocyanide, produced in the oxidation reaction, is tightly bound to the protein forming a redox couple with the heme iron. CO shifts the redox equilibrium by acting as a trap for the reduced heme. The equilibrium and kinetic aspects of the process have been accounted for in a reaction scheme where the internal electron transfer reaction is the rate-limiting step.

Determination of the rate and equilibrium constants for oxygen and carbon monoxide binding to R-state human hemoglobin cross-linked between the alpha subunits at lysine 99.

The kinetics of O2 and CO binding to R-state human hemoglobin A0 and human hemoglobin cross-linked between the alpha chains at Lys99 residues were examined using ligand displacement and partial photolysis techniques. Oxygen equilibrium curves were measured by Imai's continuous recording method (Imai, K. (1981) Methods Enzymol. 76, 438-449). The rate of the R to T transition was determined after full laser photolysis of the carbon monoxide derivative by measuring the resultant absorbance changes at an isosbestic point for ligand binding. Chemical cross-linking caused the R-state O2 affinity of alpha subunits to decrease 6-fold compared with unmodified hemoglobin. This inhibition of O2 binding was the result of both a decrease in the rate constant for ligand association and an increase in the rate constant for dissociation. The O2 affinity of R-state beta subunits was reduced 2-fold because of an increase in the O2 dissociation rate constant. These changes were attributed to proximal effects on the R-state hemes as the result of the covalent cross-link between alpha chain G helices. This proximal strain in cross-linked hemoglobin was also expressed as a 5-fold higher rate for the unliganded R to T allosteric transition. The fourth O2 equilibrium binding constant, K4, measured by kinetic techniques, could be used to analyze equilibrium curves for either native or cross-linked hemoglobin. The resultant fitted values of the Adair constants, a1, a2, and a3 were similar to those obtained when K4 was allowed to vary, and the fits were of equal quality. When K4 was fixed to the kinetically determined value, the remaining Adair constants, particularly a3, became better defined.

Lack of age-dependent changes in co binding to cardiac mitochondrial cytochrome oxidase

The rates of CO binding to cytochrome oxidase at low temperatures were studied in mitochondria isolated from the hearts of 3- and 30–32-month-old male F344/N rats. A single apparent energy of activation of 9.6 kcal/mol is observed in mitochondria from 3-month-old rats in the presence of 1% CO. In the presence of 100% CO, the energy of activation is 10.1 kcal/mol and the rate constants of CO recombination following flash photolysis are approximately twice the rate constants in the presence of 1% CO at warm temperatures indicating that an intermediate region near the heme iron can hold a maximum of two CO molecules. In 30-month-old cardiac mitochondria, recombination in the presence of 1% and 100% CO requires crossing barriers of 9.4 kcal/mol and 10.3 kca/mol height, respectively. The approximate doubling of the values of k at warm temperatures (above 225 K) indicates, as in mitochondria from young animals, that CO migration from solvent to the heme iron involves migration across two similarly sized barriers separating the Fe from an innermost intermediate region I (capable of holding only one CO) and separating region I from intermediate region I 2 (capable of holding two CO). The kinetics of CO binding to cytochrome oxidase do not change with increasing age.

Conformational substates and motions in myoglobin. External influences on structure and dynamics

Myoglobin, a simppe dioxygen-storage protein, is a good laboratory for the investigation of the connection between protein structure, dynamics, and function. Fourier-transform infrared spectroscopy on carbon-monoxymyoglobin (MbCO) shows three major CO bands. These bands are excellent probes for the investigation of the structure-function relationship. They have different CO binding kinetics and their CO dipoles form different angles with respect to the heme normal, implying that MbCO exists in three major conformational substates, A0, A1, and A3. The entropies and enthalpies of these substates depend on temperature above approximately 180 K and are influenced by pH, solvent, and pressure. These results suggest that even a protein as simple as Mb can assume a small number of clearly different structures that perform the same function, but with different rates. Moreover, protein structure and dynamics depend strongly on the interaction of the protein with its environment.

Effect of age on kinetics and carbon monoxide binding to cytochrome oxidase in synaptic and non-synaptic brain mitochondria

The kinetic parameters of cytochrome oxidase activity in synaptic and non-synaptic brain mitochondria from 3- and 30-month-old rats were determined at room temperature. The value of K m for cytochrome c increased only 12–13% with age. The maximal velocity did not change with age, but the value of V max in synaptic mitochondria is twice that observed in non-synaptic mitochondrial cytochrome oxidase. The kinetics of CO binding to cytochrome oxidase at temperatures from 183 to 225K were also studied in synaptic and non-synaptic mitochondria from 3- and 30-month-old rat forebrains. Age-dependent differences were observed only in mitochondria of synaptic origin. Following flash photolysis at low temperatures, CO migration to the iron requires crossing two free energy barriers separating two intermediate regions from the iron. In 3-month-old synaptic mitochondria, CO must migrate across a 10.3 kca/mol barrier separating two intermedite regions, I 2 and I; a 4.7 kcal/mol barrier separates the innermost region I from the iron. Each intermediate region in 3-month-old cytochrome oxidase can hold only one CO molecule. In 30-month-old synaptic mitochondria, 10.3 kcal/mol barriers separate the two intermediate regions as well as region I and the iron; each intermediate region can hold two CO molecules. Region I 2 in non-synaptic cytochrome oxidase at either age can hold two CO molecules and the innermost region I holds only one CO molecule; energy barriers of approximately 10.3 kcal/moIe separate regions I 2, I, and the iron. These age-dependent changes may reflect age-dependent conformational changes in cytochrome oxidase.

A novel mechanism of heme-heme interaction in the homodimeric hemoglobin from Scapharca inaequivalvis as manifested upon cleavage of the proximal Fe-N epsilon bond at low pH.

The CO-binding kinetics and the optical spectra of the NO derivative of the homodimeric hemoglobin from Scapharca inaequivalvis have been investigated over the range between pH 7.0 and 2.0. In the deoxygenated derivative, protonation of the proximal imidazole at very low pH values and the consequent cleavage of the Fe-N epsilon bond result in a approximately 50-fold enhancement of the rate constant for CO binding, as found in other hemoproteins. However, in the case of the hemoglobin from S. inaequivalvis, the pH profile displays a cooperative behavior (n = 1.8 +/- 0.1), a unique feature that differentiates this protein from any other hemoprotein investigated thus far. Cleavage of the proximal bond in the NO derivative of S. inaequivalvis hemoglobin likewise displays a very steep pH transition. The mode of assembly of the homodimer, in which the heme-carrying E and F helices provide the subunit interface and bring the hemes at a much shorter distance (18.4 A) than in vertebrate hemoglobins, is likely to provide the structural basis for this unique behavior.

The effects of pressure on O 2 and CO binding kinetics for hemoproteins

The effects of pressure on O 2 and CO binding kinetics for hemoproteins

Kinetic evidence for a role of heme geometry on the modulation of carbon monoxide reactivity in human hemoglobin.

Kinetics of CO binding to human hemoglobin (Hb) has been followed below neutrality. With respect to the behavior observed at pH 7.0, CO binding to deoxy-Hb at pH 2.3 displays a much faster second-order combination rate constant (1.2 x 10(-7) M-1 s-1) and loss of the autocatalytic character of the kinetic progress curve. The spectroscopic features of the transient deoxy-Hb at pH 2.75 indicate the phenomenon to be related to the cleavage of the proximal histidine N epsilon-Fe bond, as reported for monomeric hemoproteins (Coletta, M., Ascenzi, P., Traylor, T. G., and Brunori, M. (1985) J. Biol. Chem. 260, 4151-4155). The faster CO binding rate constant, higher than that characteristic of the R state, cannot be attributed to either (i) an enhanced dimerization of deoxy-Hb at low pH, or (ii) a quaternary switch of the unliganded form to the R0 state. The data indicate that interaction(s) of the heme on the proximal side is crucial in accounting for the difference in the CO binding rate constant between the two quaternary conformations of hemoglobin.

Some structural and functional properties of hemoglobin-containing liposomes (hemosomes), a potential red blood cell substitute.

There is a growing interest in the encapsulation of hemoglobin (Hb) in liposomes for two reasons: (i) the resulting hemosomes have a potential use as a non-toxic, non-immunogenic red blood cell (RBC) surrogate (1–5), and (ii) they provide a useful RBC model for studying the interaction of Hb with lipid bilayers (6,7). We prepared hemosomes by dispersing various (phospho)lipids in concentrated human RBC lysate, and report here on (i) some morphological characteristics of the resulting particles; (ii) the effect of lipid composition on the amount of entrapped Hb; (iii) the kinetics of CO-binding by entrapped Hb; and (iv) the stability of the lipid membrane and of Hb in hemosomes. To shed light on the molecular mechanism of Hb-liposome interactions, the changes in the intrinsic fluorescence of Hb upon addition to small unilamellar vesicles (SUV) were also analyzed.

Fine Tuning of Heme Reactivity: Hydrogen-Bonding and Dipole Interactions Affecting Ligand Binding to Hemoproteins

Abstract Heme reactivity in hemoproteins is governed by the microenvironment near the ligand binding site. In order to quantify polarity effects on heme ligand binding, the kinetics of O2 and CO binding have been measured for a series of synthetic heme models equipped with a range of groups of varying dipole moments positioned near the heme coordination site. For hemes with polar aprotic groups, both O2 on (k′) and off rates (k) are found to be dependent on the dipole moment. For model systems containing protic groups, the O2 off rate is substantially reduced due to hydrogen bonding with the coordinated O2. The hydrogen-bonding stabilization is estimated to be 0.7 and 1.6 kcal/mol for an alcohol and a secondary amide, respectively. CO binding displays little correlation with a polarity effect; instead it seems to depend upon the size and position of the polar group.

Xenopus laevis hemoglobin and its hybrids with hemoglobin A

Isolated alpha and beta chains from Xenopus laevis hemoglobin have been purified. The isolation procedure yields native alpha chains whose functional behavior has been characterized and compared with that of human alpha chains. Isolated beta chains in the presence of oxygen are characterized by low stability, and hence their functional characterization was limited to the CO binding kinetics. When stoichiometric amounts of the isolated alpha and beta chains are mixed, a tetramer characterized by heme-heme interactions and oxygen affinity comparable to that of the native molecule is readily reconstituted. Moreover, both chains, under appropriate conditions, form stable hybrid tetramers with the partner subunits from human hemoglobin; results on the functional properties of these hybrid hemoglobins are presented and discussed in relation to the stereochemical model of the Root effect.