Dependence of the quantum efficiency for photolysis of carboxyhemoglobin on the degree of ligation.

A combined stopped flow-laser photolysis apparatus was used to measure the quantum efficiency for removal of carbon monoxide bound to human hemoglobin as a function of fractional CO saturation. This flow-flash technique allows the properties of partially liganded hemoglobin molecules, which are sparsely populated under equilibrium conditions, to be conveniently studied. Experiments performed at pH 7 and 20 degrees C both in the presence and absence of phosphates gave a similar dependence of quantum efficiency on fractional saturation. The observed quantum efficiency was 0.90 +/- 0.06 at 10% saturation and decreased to 0.47 +/- 0.02 as full saturation was approached. An allosteric model in which Hb(CO)1 has a quantum efficiency of 0.99 while other liganded species have quantum efficiencies of 0.47 was used to produce a good simulation of the results.     

Geminate ligand recombination as a probe of the R, T equilibrium in hemoglobin

Flash photolysis kinetics of carbon monoxide hemoglobin show a decrease in the fraction of ligand recombination occurring as geminate when the hemoglobin has fewer ligands bound. Fully saturated samples, normally referred to as R state, show approximately 50% geminate phase, while samples at low saturation (T state) show less than 3%. The latter result was obtained by photolysis of samples with a short delay after stopped flow of solutions of deoxy hemoglobin (Hb) and ligand. The decrease in the fraction of geminate phase was also observed using a double flash technique. The transient mixture of R and T states generated by flash photolysis of Hb-CO was probed with a weaker time-delayed photolysis pulse. The kinetics of both the geminate and bimolecular phases following the second pulse were measured. The fraction geminate signal was least at delays where the maximum proportion of liganded T state tetramer is expected. The biphasic bimolecular process is also an indicator of the allosteric state of Hb. The populations of R and T may be determined from the overall ligand recombination kinetics; however, the analysis is model-dependent. The fraction geminate reaction may provide a rapid measure of the amount of liganded hemes in the R and T states.     

Reversible oxygenation of protoheme-imidazole complex in aqueous solution (1,2)

Solutions of protohemin in aqueous buffer containing imidazole were reduced and exposed to carbon monoxide forming the carbon monoxide-imidazole complex similar to that in carboxyhemoglobin. This complex is stable for long periods in the presence of low pressures of oxygen and thus the standard flash photolysis methods can be used to determine rates of combination of the heme-imidazole complex with oxygen. Combination rates for both carbon monoxide and oxygen are faster than any on rates for hemoglobin and oxygen dissociation rates are also faster. But the equilibrium constant for binding of this isolated site is larger than that for hemoglobin.     

Stereodynamic properties of the cooperative homodimeric Scapharca inaequivalvis hemoglobin studied through optical absorption spectroscopy and ligand rebinding kinetics.

The study of the thermal evolution of the Soret band in heme proteins has proved to be a useful tool to understand their stereodynamic properties; moreover, it enables one to relate protein matrix fluctuations and functional behavior when carried out in combination with kinetic experiments on carbon monoxide rebinding after flash photolysis. In this work, we report the thermal evolution of the Soret band of deoxy, carbonmonoxy, and nitric oxide derivatives of the cooperative homodimeric Scapharca inaequivalvis hemoglobin in the temperature range 10-300 K and the carbon monoxide rebinding kinetics after flash photolysis in the temperature range 60-200 K. The two sets of results indicate that Scapharca hemoglobin has a very rigid protein structure compared with other hemeproteins. This feature is brought out i) by the absence of nonharmonic contributions to the soft modes coupled to the Soret band in the liganded derivatives, and ii) by the almost "in plane" position of the iron atom in the photoproduct obtained approximately 10(-8) s after dissociating the bound carbon monoxide molecule at 15 K.     

The gaseous messenger carbon monoxide is released from the eye into the ophthalmic venous blood depending on the intensity of sunlight

Circadian and seasonal rhythms in daylight affect many physiological processes. In the eye, energy of intense visible light not only initiates a well-studied neural reaction in the retina that modulates the secretory function of the hypothalamus and pineal gland, but also activates the heme oxygenase (HO) to produce carbon monoxide (CO). This study was designed to determine whether the concentration of carbon monoxide (CO) in the ophthalmic venous blood changes depending on the phase of the day and differing extremely light intensity seasons: summer and winter. The concentration of CO in the venous blood flowing out from the nasal cavity, where heme oxygenase (HO) is expressed, but no photoreceptors, was used as a control. Sixteen mature males of a wild boar and pig crossbreed were used for this study. Samples of ophthalmic and nasal venous blood and systemic arterial and venous blood were collected repeatedly for two consecutive days during the longest days of the summer and the shortest days of the winter. The concentrations of CO in blood samples was measured using a standard addition method. During the longest days of the summer the concentration of CO in ophthalmic venous blood averaged 3.32 ± 0.71 and 3.43 ± 0.8 nmol/ml in the morning and afternoon, respectively, and was significantly higher than in the night averaging 0.89 ± 0.12 nmol/ml (p<0.001). During the shortest day of the winter CO concentration in ophthalmic venous blood was 1.11 ± 0.10 and 1.13 ± 0.14 nmol/ml during the light and nocturnal phase, respectively, and did not differ between phases, but was lower than in the light phase of the summer (p<0.01). The CO concentration in the control nasal venous blood did not differ between seasons and day phases and was lower than in ophthalmic venous blood during the summer (p<0.01) and winter (p<0.05). The results indicate that the gaseous messenger carbon monoxide is released from the eye into the ophthalmic venous blood depending on the intensity of sunlight.     

Evidence for a photoactivation of CO rebinding to fully reduced cytochrome c oxidase after low-temperature flash photolysis

Carbon monoxide rebinding to isolated fully reduced cytochrome c oxidase has been investigated by low-temperature, flash photolysis, dual-wavelength spectrometry. By using separately different wavelength pairs to monitor the liganding of CO to Fe a3 and by keeping all other experimental conditions identical, there has been singled out a photoactivation effect on CO rebinding. For instance, at 187 K, the rate constant of CO rebinding observed at 425-475 nm was twice that derived from the kinetic at 444-475 nm despite a rate constant of photodissociation about 10 times larger at 425-475 nm than at 444-475 nm. This new finding is discussed with respect to previous investigations under similar conditions.     

Pulsed laser flash photolysis with IR diode laser detection: the reaction of photogenerated trans-[(n-C7H16)(P(OPri)3)W(CO)4] with piperidine

Pulsed laser flash photolysis with IR detection apparatus in which the analyzing source is an IR diode laser has been used to study ligand-exchange reactions of metal carbonyl transients in solution. The apparatus is described and the advantages of the IR diode laser analyzing source over the ‘globar’ and line-tuneable CO laser sources previously employed in such studies are discussed. It has been employed to monitor the thermal reaction with pip of trans-[(n-C₇H₁₆)(L)W(CO)₄], (L = tri(isopropyl) phosphite) produced via pulsed laser flash photolysis of cis-(pip)(L)W(CO)₄ in n-heptane. This reaction obeys a mixed order rate law interpreted in terms of bimolecular ‘trapping’ of the photogenerated intermediate with pip to afford trans-(pip)(L)W(CO)₄ and a competing unimolecular pathway tentatively ascribed to slow isomerization of trans-[(L)W(CO)₄] to its cis analogue. This isomer then is rapidly trapped by pip to afford the cis-(pip)(L)W(CO)₄ photolysis precursor.     

A Spectroscopic Study of Structural Heterogeneity and Carbon Monoxide Binding in Neuroglobin

Neuroglobin (Ngb) is a small globular protein that binds diatomic ligands like oxygen, carbon monoxide (CO) and nitric oxide at a heme prosthetic group. We have performed FTIR spectroscopy in the infrared stretching bands of CO and flash photolysis with monitoring in the electronic heme absorption bands to investigate structural heterogeneity at the active site of Ngb and its effects on CO binding and migration at cryogenic temperatures. Four CO stretching bands were identified; they correspond to discrete conformations that differ in structural details and CO binding properties. Based on a comparison of bound-state and photoproduct IR spectra of the wild-type protein, Ngb distal pocket mutants and myoglobin, we have provided structural interpretations of the conformations associated with the different CO bands. We have also studied ligand migration to the primary docking site, B. Rebinding from this site is governed by very low enthalpy barriers (∼1 kJ/mol), indicating an extremely reactive heme iron. Moreover, we have observed ligand migration to a secondary docking site, C, from which CO rebinding involves higher enthalpy barriers.     

Investigation of laser-induced long-lived states of photolyzed MbCO

We present evidence from resonance Raman and absorption measurements that the extended exposure of MbCO to CW laser light at low temperatures alters the CO rebinding kinetics and leads to a significantly increased population of very long lived states of photolyzed MbCO. This optical "pumping" process is observed for samples frozen in both aqueous buffer and glycerol/buffer and exhibits power law behavior with a very weak temperature dependence. A comparison of the nonexponential rebinding kinetics of CO molecules from the pumped states with the rebinding observed in flash photolysis experiments suggests that the pumped states are distinct geminate states, not observed in flash photolysis experiments. Thus, a four-state model, with two geminate states, is implicated for MbCO. Pumped states may represent "separated geminate pair" states with the CO molecule still in the heme pocket or possibly trapped within a cavity on its way through the protein matrix, consistent with molecular dynamics simulations. The possibility of significant deoxyheme relaxation from a less domed to a more domed configuration, as a result of the multiple photolysis events associated with the pumping process, is also explored. However, the small changes observed in the Soret band line shape and position subsequent to pumping at T less than 180 K tend to rule out this explanation for the pumping process. Since the yield for creating a pumped state is small (e.g., less than 10(-7) for T greater than 100 K), pumping can be observed only after extended illumination and is absent in flash photolysis measurements, even after multiple flashes. At higher temperatures (T greater than 180 K), the escape of the CO molecule to the solvent is observed. Our data are consistent with a "phase transition" of the protein that is coupled to the surrounding matrix. The protein fluctuations are quenched below approximately 185 K for a solvent composed of 70% glycerol and below approximately 260 K for aqueous buffer. We also present the first large amplitude measurements of CO rebinding from the protein exterior, observed below 200 K after freezing the sample under laser illumination.     

Structure-dynamics-function relationships in Asian elephant (Elephas maximus) myoglobin. An optical spectroscopy and flash photolysis study on functionally important motions.

In this work we report the thermal behavior (10-300 K) of the Soret band lineshape of deoxy and carbonmonoxy derivatives of Asian elephant (Elephas maximus) and horse myoglobins together with their carbon monoxide recombination kinetics after flash photolysis; the results are compared to analogous data relative to sperm whale myoglobin. The Soret band profile is modeled as a Voigt function that accounts for the coupling with high and low frequency vibrational modes, while inhomogeneous broadening is taken into account with suitable distributions of purely electronic transition frequencies. This analysis makes it possible to isolate the various contributions to the overall lineshape that; in turn, give information on structural and dynamic properties of the systems studied. The optical spectroscopy data point out sizable differences between elephant myoglobin on one hand and horse and sperm whale myoglobins on the other. These differences, more pronounced in deoxy derivatives, involve both the structure and dynamics of the heme pocket; in particular, elephant myoglobin appears to be characterized by larger anharmonic contributions to soft modes than the other two proteins. Flash photolysis data are analyzed as sums of kinetic processes with temperature-dependent fractional amplitudes, characterized by discrete pre-exponentials and either discrete or distributed activation enthalpies. In the whole temperature range investigated the behavior of elephant myoglobin appears to be more complex than that of horse and sperm whale myoglobins, which is in agreement with the increased anharmonic contributions to soft modes found in the former protein. Thus, to satisfactorily fit the time courses for CO recombination to elephant myoglobin five distinct processes are needed, only one of which is populated over the whole temperature range investigated. The remarkable convergence and complementarity between optical spectroscopy and flash photolysis data confirms the utility of combining these two experimental techniques in order to gain new and deeper insights into the functional relevance of protein fluctuations.     

Oxygen and carbon monoxide kinetics of Glycera dibranchiata monomeric hemoglobin.

Oxygen and carbon monoxide kinetics of Glycera dibranchiata monomeric hemoglobin have been studied using laser photolysis, air flash, and stopped flow techniques. The reactions of this hemoglobin with both ligands were found to be more rapid than the corresponding reactions involving myoglobin and were also biphasic in nature, the rate constants being approximately an order of magnitude different for the fast and slow phases in each case. No pH or hemoglobin concentration dependence of the pseudo-first order rate constants was apparent between pH 6 and 9 and in the concentration range of 1.25 to 40 muM heme. Both fast and slow pseudo-first order oxygen combination rate constants varied linearly with oxygen concentration between 16 and 1300 muM. A first order slow relaxation was also noted which was linearly dependent on heme concentration and inversely dependent on oxygen concentration. This reaction has been shown to be due to a replacement of oxygen by carbon monoxide. The presence of this reaction is a result of the high affinity of Glycera monomer for carbon monoxide as shown by the partition coefficient Mr = approximately 20,000 ana an equilibrium dissociation constant of the order L = 1.1 X 10(-9) M.     

Isolation and characterization of the triply oxidized derivative of a cross-linked hemoglobin.

Hemoglobin A, cross-linked between Lys 99 alpha 1 and Lys 99 alpha 2, was used to obtain a partially oxidized tetramer in which only one of the four hemes remains reduced. Because of the absence of dimerization, asymmetric, partially oxidized derivatives are stable. This is evidenced by the fact that eight of the ten possible oxidation states could be resolved by analytical isoelectric focusing. A triply oxidized hemoglobin population HbXL+3 was isolated whose predominant component was (alpha + alpha +, beta + beta 0). This triferric preparation was examined as a possible model for the triliganded state of ferrous HbA. The aquomet and cyanomet derivatives were characterized by their CD spectra and their kinetic reactions with carbon monoxide. CD spectra in the region of 287 nm showed no apparent change in quaternary structure upon binding ligand to the fourth, ferrous heme. The spectra of the oxy and deoxy forms of the cyanomet and aquomet derivatives of HbXL+3 differed insignificantly and were characteristic of the normal liganded state. Upon addition of inositol hexaphosphate (IHP), both the oxy and deoxy derivatives of the high-spin triaquomet species converted to the native deoxy conformation. In contrast, IHP had no such effect on the conformation of the low-spin cyanomet derivatives of HbXL+3. The kinetics of CO combination as measured by stopped-flow and flash photolysis techniques present a more complex picture. In the presence of IHP the triaquomet derivative does bind CO with rate constants indicative of the T state whether these are measured by the stopped-flow technique or by flash photolysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

A polychromatic flash photolysis apparatus (PFPA)

A wide variety of biologically relevant chemical intermediates have been identified and characterised by their spectral properties. When rapid kinetics, i.e. rapid changes in these spectral properties are studied, the equipment designed for these studies (flash photolysis-, T-jump apparatus) usually allows only the registration of intensity changes of the monitoring light beam at one particular wavelength. Quite frequently, however, particularly in biological systems, the reactions of interest are too complex to be fully understood using single wavelength techniques. We have therefore designed and built a flash photolysis apparatus which permits the simultaneous recording of absorbance changes at 32 wavelengths, freely selectable between 300 and 1000 nm, as well as changes in fluorescence, luminescence, birefringence and light scattering. The apparatus, which we have called Polychromatic Flash Photolysis Apparatus (PFPA), acquires up to 8000 difference spectra per second with an amplitude resolution of better than 0.0001 absorbance unit. Data acquisition and activation of an actinic xenon flash unit occurs under computer control. The same computer is responsible for data storage, handling and graphic display. This communication describes the PFPA, its performance, and, as a demonstration of its potential usefulness, its application to the measurement of the light driven photocycle of bacterial rhodopsin, the proton pumping protein of Halobacterium halobium.     

Effect of lipids on CO recombination with ferrocytochrome P-450

Using the flash photolysis technique the kinetics of recombination of carbon monoxide with ferrocytochrome P-450 LM-2 was investigated. Ferrocytochrome P-450 was incorporated into liposomes prepared from different lipids: from microsomal lipids, phosphatidylcholine from egg yolk, dipalmitoylphosphatidylcholine. It was found that the activity and structure of ferrocytochrome P-450 conformers is affected by the lipid microenvironment. The kinetics of the CO-binding is affected also by the nature of lipids.     

Ligand binding to cytochrome c peroxidase from Pseudomonas aeruginosa

The high potential heme site of Pseudomonas cytochrome c peroxidase has His and Met as ligands. On reduction, the Fe-met bond becomes photosensitive. Following photolysis, the bond reforms with a half-time of 35 ps. The low potential heme peroxidatic site of the fully reduced enzyme has been shown to bind to a range of ligands. The compounds with carbon monoxide, methyl, ethyl, n-butyl, and t-butyl isonitriles have been investigated by laser flash photolysis. All are photosensitive and show different degrees of geminate recombination of ligand in the picosecond and nanosecond time ranges. Carbon monoxide shows the least effect. The three straight-chain isonitriles show about 50% geminate recombination with half-times of the order of 10 ns. t-Butyl isonitrile shows more and faster recombination. These results imply considerable freedom of movement within the active site for the smaller ligands.     

The kinetics of the diffusion-controlled reaction of the binding of carbon monoxide to hemoglobin in glycerol-water mixtures of high viscosity

The kinetics of the binding of carbon monoxide to human hemoglobin and to ferrous horseradish peroxidase (HRP) have been studied by flash photolysis in mixtures of glycerol and water over a wide range of temperature and solvent viscosities. This was done in order that the influence of diffusion-control on the association rates could be determined. The binding of CO to HRP which is much slower than binding to Hb was devoid of diffusion effects. By contrast, the fast and slow phases of binding to Hb in the high viscosity solvents both displayed curved Arrhenius plots, consistent with a change from a chemical activationcontrolled process in the high temperature region to a diffusion-controlled process in the low temperature region. Analyses of the curved Arrhenius plots indicated that in the low temperature diffusion-controlled region, the activation enthalpy is similar to the activation energy of viscosity of the solvent, as might be expected for a diffusion-controlled reaction.Curve fitting of rate-temperature-viscosity data, assuming simultaneous chemical activation and diffusion-control, yielded factors by which the diffusion rate constants differ from that for reaction between uniformly reactive spheres of equal radii. For the fast Hb reaction, observed upon partial photolysis, this factor varies from 0.02 to 1.1, depending upon the solvent composition. For the slow Hb reaction, observed upon higher degrees of photolysis, this factor was 0.03 and 0.04. These factors were rationalized in terms of fractional surface reactivities and of a maximum allowable solid angle of entry of reactant to the binding site. It was concluded that the steric hindrance of T-state Hb (slow reaction) is much greater than R-state Hb (fast reaction).     

Microspectrophotometry of mitochondrial cytochrome P-450 in single adrenal cells

Synopsis The cytochrome P-450 is known to be a key enzymic component in steroid hydroxylation. Biochemically it is localized in adrenal cells, both in the mitochondrial and in the microsomal fractions. Owing to its characteristic light absorptivity, attempts were made at its localization and measurement in a microspectrophotometric system. Specific difference spectra were obtained in the cytoplasm of some cells from an adrenal cell suspension before and after saturation with carbon monoxide. Scanning at 450 nm showed slender absorption maxima after CO saturation randomly distributed in the cytoplasm. These may be attributed to mitochondria or larger cytoplasmic functional units including mitochondria.     

Inhibition by carbon monoxide of the secondary-amine mono-oxygenase of Pseudomonas aminovorans and the photochemical action spectrum for its reversal.

The secondary-amine mono-oxygenase (EC 1.14.99.--) of Pseudomonas aminovorans is potently inhibited by carbon monoxide. The degree of inhibition of the purified enzyme was determined by the CO:O2 ratio rather than by the absolute concentration of carbon monoxide. The partition constant (the CO:O2 ratio causing 50% inhibition of activity) was 9.2 X 10(-4). The inhibition could be reversed by light, and the extent of reversal was proportional to the light intensity. With monochromatic light of wavelength 417 nm, the light sensitivity, L, was determined to be 2.5 X 10(8) cm2 min/mol quantum. The photochemical action spectrum for the light reversal of inhibition showed a single maximum of effectiveness at about 420 nm. The difference spectrum of the enzyme (reduced by NADH) on bubbling with CO (compared with an NADH-reduced reference sample) showed a peak at 426 nm. The preparations showed none of the spectral properties to cytochrome P-450 mono-oxygenase preparations, and was much more sensitive to carbon monoxide. The enzyme behaves as a typical o-type cytochrome (i.e. a carbon-monoxide-reactive b-type cytochrome), and its sensitivity to carbon monoxide as well as in its spectral properties, shows close resemblances to haemoglobin.     

Infrared studies of the CO-inhibited form of the Fe-only hydrogenase from Clostridium pasteurianum I: examination of its light sensitivity at cryogenic temperatures

Infrared spectroscopy has been used to examine the oxidized and CO-inhibited forms of Fe-only hydrogenase I from Clostridium pasteurianum. For the oxidized enzyme, five bands are detected in the infrared spectral region between 2100 and 1800 cm(-1). The pattern of infrared bands is consistent with the presence of two terminally coordinated carbon monoxide molecules, two terminally coordinated cyanide molecules, and one bridging carbon monoxide molecule, ligated to the Fe atoms of the active site [2Fe] subcluster. Infrared spectra of the carbon monoxide-inhibited state, prepared using both natural abundance CO and 13CO, indicate that the two terminally coordinated CO ligands that are intrinsic to the enzyme are coordinated to different Fe atoms of the active site [2Fe] subcluster. Irradiation of the CO-inhibited state at cryogenic temperatures gives rise to two species with dramatically different infrared spectra. The first species has an infrared spectrum identical to the spectrum of the oxidized enzyme, and can be assigned as arising from the photolysis of the exogenous CO from the active site. This species, which has been observed in X-ray crystallographic measurements [Lemon, B. J., and Peters, J. W. (2000) J. Am. Chem. Soc. 122, 3793], decays above 150 K. The second light-induced species decays above 80 K and is characterized by loss of the infrared band associated with the Fe bridging CO at 1809 cm(-1). Potential models for the second photolysis event are discussed.     

The glutathione thiyl radical does not react with nitrogen monoxide

Laser flash photolysis experiments shows that the rate constant for the reaction of the glutathione thiyl radical with nitrogen monoxide to give S-nitrosoglutathione is lower than 2.8+/-0.6 x 10(7)M(-1)s(-1). The conversion of the thiyl radical to its carbon-centred form at 10(3)s(-1) exceeds the formation of S-nitrosoglutathione when physiological concentrations of nitrogen monoxide are taken into account.