Sergei V. Lepeshkevich

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.

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.

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.

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 α and β 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 α and β 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 α and β subunits were revealed. Regulation of the O2‐binding properties of the α and β subunits within the liganded tetramer is proposed to be attained in two quite different ways.

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 β 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 β 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.

Laser Kinetic Spectroscopy Studies of the Bimolecular Oxygenation of α- and β-Subunits within the R-State of Human Hemoglobin

Bimolecular oxygenation of tri-liganded R-state human hemoglobin (HbA) is described by bi-exponential kinetics with association rate constants kα = 27.2 ± 1.3 (μM·sec)-1 and kβ = 62.9 ± 1.6 (μM·sec)-1. Both the observed processes have been assigned to the bimolecular oxygenation of α- and β-subunits of the native tetrameric protein by molecular oxygen. The quantum yields of photodissociation within the completely oxygenated R-state HbA are γα = 0.0120 ± 0.0017 and γβ = 0.044 ± 0.005 for α- and β-subunits, respectively. The oxygenation reactions of isolated αPCMB- and βPCMB-hemoglobin chains are described by mono-exponential kinetics with the association rate constants kα = 44 ± 2 (μM·sec)-1 and kβ = 51 ± 1 (μM·sec)-1, respectively. The quantum yields of photodissociation of isolated αPCMB- and βPCMB-chains (0.056 ± 0.006 and 0.065 ± 0.006, respectively) are greater than that observed for appropriate subunits within the R-state of oxygenated HbA.