Towards understanding non-equivalence of α and ß subunits within human hemoglobin in conformational relaxation and molecular oxygen rebinding.

Picosecond to millisecond laser time-resolved transient absorption spectroscopy was used to study molecular oxygen (O2) rebinding and conformational relaxation following O2 photodissociation in the α and ß subunits within human hemoglobin in the quaternary R-like structure. Oxy-cyanomet valency hybrids, α2(Fe2+-O2)ß2(Fe3+-CN) and α2(Fe3+-CN)ß2(Fe2+-O2), were used as models for oxygenated R-state hemoglobin. An extended kinetic model for geminate O2 rebinding in the ferrous hemoglobin subunits, ligand migration between the primary and secondary docking site(s), and nonexponential tertiary relaxation within the R quaternary structure, was introduced and discussed. Significant functional non-equivalence of the α and ß subunits in both the geminate O2 rebinding and concomitant structural relaxation was revealed. For the ß subunits, the rate constant for the geminate O2 rebinding to the unrelaxed tertiary structure and the tertiary transition rate were found to be greater than the corresponding values for the α subunits. The conformational relaxation following the O2 photodissociation in the α and ß subunits was found to decrease the rate constant for the geminate O2 rebinding, this effect being more than one order of magnitude greater for the ß subunits than for the α subunits. Evidence was provided for the modulation of the O2 rebinding to the individual α and ß subunits within human hemoglobin in the R-state structure by the intrinsic heme reactivity through a change in proximal constraints upon the relaxation of the tertiary structure on a picosecond to microsecond time scale. Our results demonstrate that, for native R-state oxyhemoglobin, O2 rebinding properties and spectral changes following the O2 photodissociation can be adequately described as the sum of those for the α and ß subunits within the valency hybrids. The isolated ß chains (hemoglobin H) show similar behavior to the ß subunits within the valency hybrids and can be used as a model for the ß subunits within the R-state oxyhemoglobin. At the same time, the isolated α chains behave differently to the α subunits within the valency hybrids.

Molecular oxygen migration through the xenon docking sites of human hemoglobin in the R-state.

A nanosecond laser flash-photolysis technique was used to study bimolecular and geminate molecular oxygen (O2) rebinding to tetrameric human hemoglobin and its isolated α and ß chains in buffer solutions equilibrated with 1atm of air and up to 25atm of xenon. Xenon binding to the isolated α chains and to the α subunits within tetrameric hemoglobin was found to cause a decrease in the efficiency of O2 escape by a factor of ~1.30 and 3.3, respectively. A kinetic model for O2 dissociation, rebinding, and migration through two alternative pathways in the hemoglobin subunits was introduced and discussed. It was shown that, in the isolated α chains and α subunits within tetrameric hemoglobin, nearly one- and two-third escaping molecules of O2 leave the protein via xenon docking sites, respectively. The present experimental data support the idea that O2 molecule escapes from the ß subunits mainly through the His(E7) gate, and show unambiguously that, in the α subunits, in addition to the direct E7 channel, there is at least one alternative escape route leading to the exterior via the xenon docking sites.

Photosensitized singlet oxygen luminescence from the protein matrix of Zn-substituted myoglobin.

A nanosecond laser near-infrared spectrometer was used to study singlet oxygen ((1)O2) emission in a protein matrix. Myoglobin in which the intact heme is substituted by Zn-protoporphyrin IX (ZnPP) was employed. Every collision of ground state molecular oxygen with ZnPP in the excited triplet state results in (1)O2 generation within the protein matrix. The quantum yield of (1)O2 generation was found to be equal to 0.9 ± 0.1. On the average, six from every 10 (1)O2 molecules succeed in escaping from the protein matrix into the solvent. A kinetic model for (1)O2 generation within the protein matrix and for a subsequent (1)O2 deactivation was introduced and discussed. Rate constants for radiative and nonradiative (1)O2 deactivation within the protein were determined. The first-order radiative rate constant for (1)O2 deactivation within the protein was found to be 8.1 ± 1.3 times larger than the one in aqueous solutions, indicating the strong influence of the protein matrix on the radiative (1)O2 deactivation. Collisions of singlet oxygen with each protein amino acid and ZnPP were assumed to contribute independently to the observed radiative as well as nonradiative rate constants.

Does photodissociation of molecular oxygen from myoglobin and hemoglobin yield singlet oxygen?

Time-resolved luminescence measurements in the near-infrared region indicate that photodissociation of molecular oxygen from myoglobin and hemoglobin does not produce detectable quantities of singlet oxygen. A simple and highly sensitive method of luminescence quantification is developed and used to determine the upper limit for the quantum yield of singlet oxygen production. The proposed method was preliminarily evaluated using model data sets and confirmed with experimental data for aqueous solutions of 5,10,15,20-tetrakis(4-N-methylpyridyl) porphyrin. A general procedure for error estimation is suggested. The method is shown to provide a determination of the integral luminescence intensity in a wide range of values even for kinetics with extremely low signal-to-noise ratio. The present experimental data do not deny the possibility of singlet oxygen generation during the photodissociation of molecular oxygen from myoglobin and hemoglobin. However, the photodissociation is not efficient to yield singlet oxygen escaped from the proteins into the surrounding medium. The upper limits for the quantum yields of singlet oxygen production in the surrounding medium after the photodissociation for oxyhemoglobin and oxymyoglobin do not exceed 3.4×10(-3) and 2.3×10(-3), respectively. On the average, no more than one molecule of singlet oxygen from every hundred photodissociated oxygen molecules can succeed in escaping from the protein matrix.

The kinetics of molecular oxygen migration in the isolated α chains of human hemoglobin as revealed by molecular dynamics simulations and laser kinetic spectroscopy.

Bimolecular and germinate molecular oxygen (O(2)) rebinding to isolated α chains of human adult hemoglobin in solutions is analyzed. Multiple extended molecular dynamics (MD) simulations of the O(2) migration within the protein after dissociation are described. Computational modeling is exploited to identify hydrophobic pockets within the αchains and internal O(2) migration pathways associated with the experimentally observed ligand rebinding kinetics. To initiate dissociation, trajectories of the liganded protein are interrupted, the iron-dioxygen bond is broken, and the parameters of the iron-nitrogen bonds are simultaneously altered to produce a deoxyheme conformation. MD simulations provide 140 essentially independent trajectories (up to 25-ns long) of the O(2) migration in the protein. The time dependence of cavities occupancy, obtained by the MD simulations, and the kinetics of O(2) rebinding, measured by flash-photolysis techniques, allow us to obtain the kinetics of the entire O(2) migration process within the nanosecond time range and construct an explicit kinetic model of the O(2) migration and rebinding process. The amino acids that have the most pronounced effect on the ligand migration within the α chain matrix are predicted.

Molecular oxygen binding with alpha and beta subunits within the R quaternary state of human hemoglobin in solutions and porous sol-gel matrices.

Nanosecond laser flash-photolysis technique was used to study bimolecular and geminate molecular oxygen (O2) rebinding to alpha and beta subunits within oxygenated human adult hemoglobin in solutions and porous wet sol-gel matrices. Plasticity associated with the tertiary structure within R-state hemoglobin is explored through measurements that focus on the functional properties of hemoglobin under conditions designed to tune the tertiary structure without inducing the R to T transition. Inequivalence in the O2 binding to the alpha and beta hemes within the R quaternary structure is studied. The individual kinetic properties of the alpha and beta subunits within the hemoglobin encapsulated in sol-gels and aged as the oxy derivative are shown to be independent of proton concentration over the pH range from 6.3 to 8.5. However, buffer effects on the subunits' properties are revealed in sol-gel-free mediums. Interestingly, the alpha and beta subunits within the encapsulated hemoglobin possess the O2 rebinding properties which fall within the range of the ones for oxygenated hemoglobin in the buffer solutions. The combined results show a pattern in which there is a progression of functional properties that are ascribed to a family of conformational substates of R-state hemoglobin. O2 rebinding to the alpha and beta subunits within the oxygenated R-state hemoglobin in both solutions and wet sol-gels is revealed to be modulated by tertiary structural changes in two quite different ways. The possible structural changes, which modify the O2 rebinding properties, are discussed.

Effect of zinc and cadmium ions on structure and function of myoglobin.

Laser flash photolysis technique was used to study zinc and cadmium ion effects on bimolecular and nanosecond geminate molecular oxygen (O(2)) rebinding to horse heart myoglobin. Time courses for geminate recombination are analyzed in terms of a three-step, side path model. In the presence of metal ions, the greatest changes are observed in the rate constant of the O(2) rebinding from within the primary docking site and the rate constant of the O(2) migration from the primary site to the secondary xenon docking sites. The study revealed that modulation of the myoglobin affinity for O(2) by zinc and cadmium occurs at the level of the innermost barrier controlling O(2) rebinding from within the primary docking site. Sets of the calculated rate constants provide a basis for an interpretation of metal ion effects on the myoglobin structure. Overall, the results demonstrate that the metal ions binding to myoglobin gives rise to an increase in the population of the "open" distal pocket protein conformation.

Mutual effects of proton and sodium chloride on oxygenation of liganded human hemoglobin.

The different effects of pH and NaCl on individual O2-binding properties of alpha and beta subunits within liganded tetramer and dimer of human hemoglobin (HbA) were examined in a number of laser time-resolved spectroscopic measurements. A previously proposed approach [Dzhagarov BM & Lepeshkevich SV (2004) Chem Phys Lett390, 59-64] was used to determine the extent of subunit dissociation rate constant difference and subunit affinity difference from a single flash photolysis experiment. To investigate the effect of NaCl concentration on the association and dissociation rate constants we carried out a series of experiments at four different concentrations (0.1, 0.5, 1.0 and 2.0 m NaCl) over the pH range of the alkaline Bohr effect. As the data suggest, the individual properties of the alpha and beta subunits within the completely liganded tetrameric hemoglobin did not depend on pH under salt-free conditions. However, different effects NaCl on the individual kinetic properties of the alpha and beta subunits were revealed. Regulation of the O2-binding properties of the alpha and beta subunits within the liganded tetramer is proposed to be attained in two quite different ways.

A kinetic description of dioxygen motion within alpha- and beta-subunits of human hemoglobin in the R-state: geminate and bimolecular stages of the oxygenation reaction.

Laser flash photolysis technique is used to study human hemoglobin (HbA) oxygenation. Monomolecular geminate oxygenation of triliganded R-state HbA molecules is described by a function of three exponentials. Geminate oxygenation of the alpha-subunit within R-state HbA is characterized by two components with time constants of 0.14 and 1 ns, while geminate oxygenation of the beta-subunit within HbA is characterized by two components with time constants of 1 and approximately 30 ns. Bimolecular oxygenation of triliganded R-state HbA molecules is described by a biexponential law. Two observed rate constants are assigned to oxygenation of the alpha- and beta-subunit within HbA. The bimolecular association rate constants for O(2) rebinding with the alpha- and beta-subunit within triliganded R-state HbA are k(alpha) = 18.8 +/- 1.3 (microM x s)(-1) and k(beta) = 52 +/- 4 (microM x s)(-1), respectively. The apparent quantum yields of photodissociation of the beta- and alpha-subunit within completely oxygenated R-state HbA differ from each other by a factor of 3.6 and are equal to 0.041 +/- 0.004 and 0.0114 +/- 0.0012, respectively. The apparent quantum yield of photodissociation of completely oxygenated R-state HbA is equal to 0.026 +/- 0.003.