Kinetic hole burning, hole filling, and conformational relaxation in heme proteins: direct evidence for the functional significance of a hierarchy of dynamical processes

Band III is a disorder and conformation-sensitive near-infrared (approximately 760 nm) charge transfer absorption band characteristic of equilibrium and nonequilibrium five coordinate ferrous high-spin hemes. The time evolution of this absorption band subsequent to photodissociation of six coordinate ferrous hemoglobin or myoglobin can provide detailed information regarding conformational relaxation, including the thermally driven fluctuations that result in the transition from inhomogeneous to homogeneous ligand rebinding kinetic. Such time-resolved measurements over a range of temperatures are difficult due to long sample recovery times at cryogenic temperatures. A new restoring technique that allows for the rapid movement of a large optically accessible cryostat is used in combination with nanosecond time-resolved near-infrared absorption spectroscopy to generate band III as a function of time for the photoproducts of the carbon monoxide derivative of adult human hemoglobin (COHbA) and, to a more limited extent, horse myoglobin (COMb). The measurements are made over a wide range of temperatures extending from well below the solvent (75% glycerol:water) glass transition at approximately 180 K to ambient temperatures. Three temperature- and/or viscosity-dependent phenomena are observed. At the highest temperatures, only conformational relaxation is observed for the 75% glycerol sample. At very high viscosity (> or = 400 cp), conformational relaxation slows dramatically, and both kinetic hole burning followed by the filling in of the "hole" (dynamic hole filling) are observed. As the temperature is lowered, conformational relaxation slows and finally ceases. Kinetic hole burning and dynamic hole filling as well as additional broadening of band III are observed down to 140 K. The observation of kinetic hole burning (KHB) is indicative of the sample being inhomogeneous on the time scale of the ligand rebinding giving rise to KHB. The onset of hole filling is a direct manifestation of the thermal homogenization of the initial inhomogeneous distribution of conformational substates responsible for KHB. The observed dynamics are used to explain the inverse temperature effect associated with the non-Arrhenius slow down of geminate rebinding above approximately 180 K. The inverse temperature effect appears to arise not only from the onset of conformational relaxation but also from the increase in the rate on thermal averaging of the initial inhomogeneous distribution of conformational substates.     

Are smokers' self-reports of inhalation a useful measure of smoke exposure

The relation between self-assessed and objective measures of inhalation was studied in 75 smokers who assigned themselves to one of four inhalation categories, and also estimated inhalation using a rating scale. The analysis of presmoking carbon monoxide concentration in expired air, and of the rise in carbon monoxide concentration over smoking, provided an objective measure of inhalation. These was a weak but significant correlation between self-rated inhalation and rise in carbon monoxide, but no correlation with the longer-term exposure measured by presmoking levels of carbon monoxide. Differences in exposure to carbon monoxide according to self-assessed inhalation category were non-significant. It is concluded that neither subjective measure of inhalation contributes usefully to the estimation of smoke exposure among smokers who inhale.     

Deuterium off-resonance rotating frame spin-lattice relaxation of macromolecular bound ligands

Deuterated 3-trimethylsilylpropionic acid binding to bovine serum albumin was used as a model system to examine the feasibility and limitations of using the deuterium off-resonance rotating frame spin-lattice relaxation experiment for the study of equilibrium ligand-binding behavior to proteins. The results of this study demonstrate that the rotational-diffusion behavior of the bound species can be monitored directly, i.e., the observed correlation time of the ligand in the presence of a protein is approximately equal to the correlation time of the ligand in the bound state, provided that the fraction of bound ligand is at least 0.20. The presence of local ligand motion and/or chemical exchange contributions to relaxation in the bound state was inferred from the observation that the correlation time of the bound ligand was somewhat smaller than the correlation time characterizing the overall tumbling of the protein. An approximate value for the fraction of bound ligand was obtained from off-resonance relaxation experiments when supplemental spin-lattice or transverse relaxation times were employed in the analysis. Incorporation of local motion effects for the bound species into the theoretical relaxation formalism enabled the evaluation of an order parameter and an effective correlation time, which in conjunction with a wobbling in a cone model, provided additional information about ligand motion in the bound state.     

Circular dichroism spectroscopy of Lucina I hemoglobin

The monomeric hemoglobin from the mollusc Lucina pectinata (HbI) represents an interesting model system for the study of heme-related circular dichroic (CD) bands in view of the highly asymmetric distribution of aromatic residues around the heme pocket revealed by the X-ray crystal structure. The CD spectra of both ferrous and ferric HbI derivatives exhibit negative CD bands in the Soret and ultraviolet region with an enhanced ellipticity of the heme N and L bands in the near-UV region. In contrast, the magnitude of the Cotton effect in the visible and Soret regions is comparable to that observed in other hemoproteins. The spectrum of the carbon monoxide derivative shows a surprising similarity with that observed for the soybean leghemoglobin carbon monoxide adduct. A common structural feature in the two proteins is the presence in the distal pocket of two Phe residues (B9 and B10) the aromatic rings of which are perpendicular to the heme plane.     

The association between ambient carbon monoxide levels and daily mortality in Toronto, Canada

The role of ambient levels of carbon monoxide (CO) in the exacerbation of heart problems in individuals with both cardiac and other diseases was examined by comparing daily variations in CO levels and daily fluctuations in nonaccidental mortality in metropolitan Toronto for the 15-year period 1980-1994. After adjusting the mortality time series for day-of-the-week effects, nonparametic smoothed functions of day of study and weather variables, statistically significant positive associations were observed between daily fluctuations in mortality and ambient levels of carbon monoxide, nitrogen dioxide, sulfur dioxide, coefficient of haze, total suspended particulate matter, sulfates, and estimated PM2.5 and PM10. However, the effects of this complex mixture of air pollutants could be almost completely explained by the levels of CO and total suspended particulates (TSP). Of the 40 daily nonaccidental deaths in metropolitan Toronto, 4.7% (95% confidence interval of 3.4%-6.1%) could be attributable to CO while TSP contributed an additional 1.0% (95% confidence interval of 0.2-1.9%), based on changes in CO and TSP equivalent to their average concentrations. Statistically significant positive associations were observed between CO and mortality in all seasons, age, and disease groupings examined. Carbon monoxide should be considered as a potential public health risk to urban populations at current ambient exposure levels.     

Theoretical study of the distal-side steric and electrostatic effects on the vibrational characteristics of the FeCO unit of the carbonylheme proteins and their models

The vibronic theory of activation and quantum chemical intermediate neglect of differential overlap (INDO) calculations are used to study the activation of carbon monoxide (change of the C-O bond index and force field constant) by the imidazole complex with heme in dependence on the distortion of the porphyrin ring, geometry of the CO coordination, iron-carbon and iron-imidazole distances, iron displacement out of the porphyrin plane, and presence of the charged groups in the heme environment. It is shown that the main contribution to the CO activation stems from the change in the sigma donation from the 5 sigma CO orbital to iron, and back-bonding from the iron to the 2 pi orbital of CO. It follows from the results that none of the studied distortions can explain, by itself, the wide variation of the C-O vibrational frequency in the experimentally studied model compounds and heme proteins. To study the dependence of the properties of the FeCO unit on the presence of charged groups in the heme environment, the latter are simulated by the homogeneous electric field and point charges of different magnitude and location. The results show that charged groups can strongly affect the strength of the C-O bond and its vibrational frequency. It is found that the charges located on the distal side of the heme plane can affect the Fe-C and C-O bond indexes (and, consequently, the Fe-C and C-O vibrational frequencies), both in the same and in opposite directions, depending on their position. The theoretical results allow us to understand the peculiarities of the effect of charged groups on the properties of the FeCO unit both in heme proteins and in their model compounds.     

Step-scan time-resolved FTIR spectroscopy of cytochrome P-450cam carbon monoxide complex: a salt link involved in the ligand-rebinding process

Step-scan time-resolved Fourier transform infrared spectroscopy with a time resolution of 5 micros was applied to the carbon monoxide complex of cytochrome P-450cam (CYP101) to study the bimolecular ligand-rebinding process after flash photolysis. Spectral changes in the CO ligand stretch vibration band and in the protein amide I' band were monitored simultaneously. In substrate complexes having the camphor C-8, C-9, and C-10 methyl groups, rebinding of the ligand and the relaxation of the protein proceed at the same rate within experimental errors. For substrate complexes missing the methyl groups, the relaxation fo the protein tends to relax slightly faster than the CO ligand rebinding to the heme iron. compared to the (1R)-camphor and the camphane complex, the bimolecular rebinding rate constant for P-450 bound with substrates lacking the methyl groups are increased by a factor of 10-40. An unusual signal at about 1719 cm-1 was found in the difference spectrum of the photolyzed minus nonphotolyzed CO complex which has not ben reported for other heme proteins so far. This signal is strongly pronounced in wild-type P-450cam bound with (1R)-camphor or camphane and in the D251N mutant bound with (1R)-camphor. In contrast, substrate-free P-450 and the norbornane and norcamphor complexes reveal only a very weak signal or a changed band shape. On the basis of the crystal structure data, we suggest that this signal originates from the rearrangement of the hydrogen-bonding pattern or the protonation state of the salt link between Asp297, Arg299, and the heme propionate group.     

The apolar distal histidine mutant (His69-->Val) of the homodimeric Scapharca hemoglobin is in an R-like conformation

The effect of the apolar mutation of the distal histidine (His69-->Val) has been studied in the cooperative homodimeric hemoglobin from the mollusc Scapharca inaequivalvis. Absorption, circular dichroism, and resonance Raman spectroscopy point to a more symmetric heme structure of the deoxy derivative, which is indicative of an R-like conformation of the deoxy heme. Resonance Raman spectroscopy also brings out alterations in the geometry and interactions of the bound CO molecule. The iron-carbon stretching frequency is decreased by about 30 cm-1 with respect to the native protein, while the diatomic ligand stretching frequency is increased by about the same degree. Consistent with the structural changes, the ligand binding properties are significantly altered. In the mutant the overall rate and the affinity for CO binding are increased about 100-fold with respect to the native protein, and cooperativity is abolished. In addition, the amplitude and the rate of the geminate rebinding process increase significantly. This finding may be correlated to the longer average residence time of the photolyzed CO molecule within the heme pocket of the H69V mutant, as indicated by molecular dynamics simulations.     

Placental diffusing capacities at varied carbon monoxide tensions

To test the hypothesis that carbon monoxide transfer across the placenta is, in part, a facilitated process, we have looked for evidence of saturation kinetics for carbon monoxide. In eight pregnant ewes, fetal to maternal carbon monoxide transfer was examined in a preparation in which the fetal side of the placenta was perfused with blood. The carboxyhemoglobin concentrations on the fetal side of the placenta were varied from 4.8 to 70% in 23 measurements. At increased carbon monoxide tensions, the transfer from fetus to mother always decreased. The slope of log rate of carbon monoxide transfer vs. log partial pressure gradient across the placenta was significantly different from 1. Placental membrane diffusing capacity was calculated separately from total placental diffusing capacity which includes hemoglobin reaction rates and erythrocyte membrane diffusion. Placental membrane diffusing capacity decreased at increased carbon monoxide tensions. Placental permeability for urea did not change with increasing carbon monoxide tensions. These results are consistent with the hypothesis that carbon monoxide diffusion in the placenta is, in part, carrier mediated.     

Fourier transform infrared evidence for connectivity between CuB and glutamic acid 286 in cytochrome bo3 from Escherichia coli

Photodissociation of fully reduced, carbonmonoxy cytochrome bo3 causes ultrafast transfer of carbon monoxide (C triple bond O) from heme iron to CuB in the binuclear site. At low temperatures, the C triple bond O remains bound to CuB for extended times. Here, we show that the binding of C triple bond O to CuB perturbs the IR stretch of an un-ionized carboxylic acid residue, which is identified as Glu286 by mutation to Asp or to Cys. Before photodissociation, the carbonyl (C=O)-stretching frequency of this carboxylic acid residue is 1726 cm-1 for Glu286 and 1759 cm-1 for Glu286Asp. These frequencies are definitive evidence for un-ionized R-COOH and suggest that the carboxylic acids are hydrogen-bonded, though more extensively in Glu286. In Glu286Cys, this IR feature is lost altogether. We ascribe the frequency shifts in the C=O IR absorptions to the effects of binding photodissociated C triple bond O to CuB, which are relay ed to the 286 locus. Conversely, the 2065 cm-1 C triple bond O stretch of CuB-CO is markedly affected by both mutations. These effects are ascribed to changes in the Lewis acidity of CuB, or to displacement of a CuB histidine ligand by C triple bond O. C triple bond O binding to CuB also induces a downshift of an IR band which can be attributed to an aromatic C-H stretch, possibly of histidine imidazole, at about 3140 cm-1. The results suggest an easily polarizable, through-bond connectivity between one of the histidine CuB ligands and the carboxylic group of Glu286. A chain of bound water molecules may provide such a connection, which is of interest in the context of the proton pump mechanism of the heme-copper oxidases.     

Functional heterogeneity of the alpha and beta subunits in the association reaction between hemoglobin and carbon monoxide

A technique is described for the rapid inactivation and removal of excess ferricyanide used for the non-cryogenic oxidation of the unliganded subunits of the intermediates in the association reaction between hemoglobin and carbon monoxide. Under these conditions the asymmetric oxidized intermediates, which dissociate into non-identical dimers, disproportionate into their parent tetramers and four species, Hb+, HbCO, alpha 2+ beta 2CO, alpha 2CO beta 2+, are isolated by non-cryogenic isoelectric focusing. The relative concentrations of species alpha 2CO beta 2+ and alpha 2+ beta 2CO measure the overall distribution of the ligand between the alpha and beta subunits in the association reaction. At 20 degrees C in 0.1 M KCl, pH 7, preferential CO binding to the beta subunits was observed, in agreement with observations made by the cryogenic technique for the isolation of the intermediates [M. Perrella, N. Davids and L. Rossi-Bernardi, J. Biol. Chem. 267 (1992) 8744].     

Long chain W3 polyunsaturated fatty acids and lipid pattern in the mother and the newborn infant

The ratio of oxygen to carbon monoxide binding to the three fully saturated human embryonic hemoglobins has been determined. The embryonic hemoglobins exhibit significantly lower values of carbon monoxide binding than any previously reported mammalian fetal or adult hemoglobins. The possible protective role of this with regards to carbon monoxide intoxication is discussed. These data are combined with previous parameters yielding a significant correlation between oxygen affinity and carbon monoxide binding. A possible molecular origin of this correlation is discussed.     

A comparison of functional and structural consequences of the tyrosine B10 and glutamine E7 motifs in two invertebrate hemoglobins (Ascaris suum and Lucina pectinata)

The architecture of the distal heme pocket in hemoglobins and myoglobins can play an important role in controlling ligand binding dynamics. The size and polarity of the residues occupying the distal pocket may contribute steric and dielectric effects. In vertebrate systems, the distal pocket typically contains a "distal" histidine at position E7 and a leucine at position B10. There are several invertebrate organisms that have hemoglobins or myoglobins that display a pattern in which residues E7 and B10 are a glutamine and tyrosine, respectively. These proteins often have very high oxygen affinities stemming from very slow ligand off rates. In this study, two such hemoglobins, one from the nematode Ascaris suum and the other from the sulfide-fixing clam Lucina pectinata, are compared with respect to conformational and functional properties. Ultraviolet resonance Raman spectroscopy and visible resonance Raman spectroscopy are used to probe, respectively, the ligand-dependent hydrogen bonding pattern of the tyrosine residues and the proximal heme pocket interactions. Fourier transform infrared absorption spectroscopy is used to probe the dielectric properties of the distal heme pocket through the stretching frequency of carbon monoxide bound to the heme. Functionality is probed through the geminate rebinding of both CO and O2. The findings reveal two very different patterns indicative of two different mechanisms for achieving low oxygen off rates. In Hb Ascaris, a hydrogen bonding network that includes the E7 Gln, B10 Tyr, and oxygen bound to the heme results in a tight cage for the oxygen. Dissociation of the O2 requires a large amplitude conformational fluctuation that results both in a spontaneous dissociation of the oxygen through the loss of hydrogen bond stabilization and in an enhanced probability for ligand escape though the transient disruption and opening of the tight distal cage. In the case of the Hb from Lucina, there is no evidence for a tight cage. Instead the data support a model in which the hydrogen bonding network is far more tenuous and the equilibrium state of distal pocket is far more open and accessible than is the case in Ascaris. The results explain why Hb Ascaris has one of the highest oxygen affinities known (P50 approximately 10(-)3 Torr) while Hb Lucina II has an oxygen affinity comparable to that of Mb (P50 = 0.13 Torr) even though both of these Hbs contain the B10 Tyr and E7 Gln motif and display very low oxygen off rates. The roles of water and proximal strain are discussed.     

Mutation of residue Phe97 to Leu disrupts the central allosteric pathway in Scapharca dimeric hemoglobin

Residue Phe97, which is thought to play a central role in the cooperative functioning of Scapharca dimeric hemoglobin, has been mutated to leucine to test its proposed role in mediating cooperative oxygen binding. This results in an 8-fold increase in oxygen affinity and a marked decrease in cooperativity. Kinetic measurements of ligand binding to the Leu97 mutant suggest an altered unliganded (deoxy) state, which has been confirmed by high resolution crystal structures in the unliganded and carbon monoxide-liganded states. Analysis of the structures at allosteric end points reveals them to be remarkably similar to the corresponding wild-type structures, with differences confined to the disposition of residue 97 side chain, F-helix geometry, and the interface water structure. Increased oxygen affinity results from the absence of the Phe97 side chain, whose tight packing in the heme pocket of the deoxy state normally restricts the heme from assuming a high affinity conformation. The absence of the Phe97 side chain is also associated with diminished cooperativity, since Leu97 packs in the heme pocket in both states. Residual cooperativity appears to be coupled with observed structural transitions and suggests that parallel pathways for communication exist in Scapharca dimeric hemoglobin.     

Prognostic risk factors in low stage testicular germ cell tumors: unanswered questions regarding clinically useful prognosticators for extratesticular disease

Carbon monoxide is hypothesized to be produced by the enzyme heme oxygenase predominantly in liver and spleen, bound to hemoglobin, and excreted by the lungs. Thus, venous carboxyhemoglobin is expected to be higher or equal to arterial carboxyhemoglobin. Unspecific inflammatory stimuli have been shown to induce heme oxygenase in lung tissue possibly leading to pulmonary carbon monoxide production. Arterial and central venous carboxyhemoglobin levels were measured in critically ill patients on the third day of ICU stay (n = 59) as well as in otherwise healthy humans prior to orthopedic surgery (n = 29). Arterial and central venous carboxyhemoglobin were higher in ICU patients than in healthy humans, respectively. In both groups, arterial carboxyhemoglobin was significantly higher than central venous carboxyhemoglobin. The arteriovenous carboxyhemoglobin differences were similar in both groups. The data suggest (a) increased CO-generation in critical illness and (b) pulmonary CO-production in healthy and critically ill humans.     

Low frequency vibrational modes in proteins: changes induced by point-mutations in the protein-cofactor matrix of bacterial reaction centers

As a step toward understanding their functional role, the low frequency vibrational motions (<300 cm-1) that are coupled to optical excitation of the primary donor bacteriochlorophyll cofactors in the reaction center from Rhodobacter sphaeroides were investigated. The pattern of hydrogen-bonding interaction between these bacteriochlorophylls and the surrounding protein was altered in several ways by mutation of single amino acids. The spectrum of low frequency vibrational modes identified by femtosecond coherence spectroscopy varied strongly between the different reaction center complexes, including between different mutants where the pattern of hydrogen bonds was the same. It is argued that these variations are primarily due to changes in the nature of the individual modes, rather than to changes in the charge distribution in the electronic states involved in the optical excitation. Pronounced effects of point mutations on the low frequency vibrational modes active in a protein-cofactor system have not been reported previously. The changes in frequency observed indicate a strong involvement of the protein in these nuclear motions and demonstrate that the protein matrix can increase or decrease the fluctuations of the cofactor along specific directions.     

Direct observation of cooling of heme upon photodissociation of carbonmonoxy myoglobin

The formation of vibrationally excited heme upon photodissociation of carbonmonoxy myoglobin and its subsequent vibrational energy relaxation was monitored by picosecond anti-Stokes resonance Raman spectroscopy. The anti-Stokes intensity of the nu4 band showed immediate generation of vibrationally excited hemes and biphasic decay of the excited populations. The best fit to double exponentials gave time constants of 1.9 +/- 0.6 and 16 +/- 9 picoseconds for vibrational population decay and 3.0 +/- 1.0 and 25 +/- 14 picoseconds for temperature relaxation of the photolyzed heme when a Boltzmann distribution was assumed. The decay of the nu4 anti-Stokes intensity was accompanied by narrowing and frequency upshift of the Stokes counterpart. This direct monitoring of the cooling dynamics of the heme cofactor within the globin matrix allows the characterization of the vibrational energy flow through the protein moiety and to the water bath.     

Two-dimensional distributions of activation enthalpy and entropy from kinetics by the maximum entropy method

The maximum entropy method (MEM) is used to numerically invert the kinetics of ligand rebinding at low temperatures to obtain the underlying two-dimensional distribution of activation enthalpies and entropies, g(H,S). A global analysis of the rebinding of carbon monoxide (CO) to myoglobin (Mb), monitored in the Soret band at temperatures from 60 to 150 K, is performed using a Newton-Raphson optimization algorithm. The MEM approach describes the data much better than traditional least-squares analyses, reducing chi 2 by an order of magnitude. The MEM resolves two barrier distributions suggestive of rebinding to different bound conformations of MbCO, the so-called A1 and A3 substates, whose activation barriers have been independently estimated from kinetics monitored in the infrared. The distribution corresponding to A3 possesses higher activation entropies, also consistent with infrared measurements. Within an A substate, correlations of S and H are recovered qualitatively from simulated data but can be difficult to obtain from experimental data. When the rebinding measured at 60 K is excluded from the inversion, two peaks are no longer clearly resolved. Thus, data of very high quality are required to unambiguously determine the kinetic resolvability of subpopulations and the shape of the barrier distribution for a single A substate.     

Dynamics of fluorescence marker concentration as a probe of mobility

We have developed an effective experimental system for the characterization of molecular and structural mobility. It incorporates a modified fluorescence microscope geometry and a variety of analytical techniques to measure effective diffusion coefficients ranging over almost six orders of magnitude, from less than 10(-11) cm2/s to greater than 10(-6) cm2/s. Two principal techniques, fluorescence correlation spectroscopy (FCS) and fluorescence photobleaching recovery (FPR), are employed. In the FPR technique, translational transport rates are measured by monitoring the evolution of a spatial inhomogeneity of fluorescence that is produced photochemically in a microscopic volume by a short burst of intense laser radiation. In contrast, FCS uses laser-induced fluorescence to probe the spontaneous concentration fluctuations in microscopic sample volumes. The kinetics are analyzed by computing time-correlation functions of the stochastic fluctuations of the measured fluorescence intensity. The optical system and digital photocount correlator designed around a dedicated minicomputer are described and discussed. The general power of these techniques is demonstrated with examples from studies conducted on bulk solutions, lipid bilayer membranes, and mammalian cell plasma membranes.