Abstract
CO recombination kinetics has been investigated in the type II truncated hemoglobin from Thermobifida fusca (Tf-trHb) over more than 10 time decades (from 1 ps to ∼100 ms) by combining femtosecond transient absorption, nanosecond laser flash photolysis and optoacoustic spectroscopy. Photolysis is followed by a rapid geminate recombination with a time constant of ∼2 ns representing almost 60% of the overall reaction. An additional, small amplitude geminate recombination was identified at ∼100 ns. Finally, CO pressure dependent measurements brought out the presence of two transient species in the second order rebinding phase, with time constants ranging from ∼3 to ∼100 ms. The available experimental evidence suggests that the two transients are due to the presence of two conformations which do not interconvert within the time frame of the experiment. Computational studies revealed that the plasticity of protein structure is able to define a branched pathway connecting the ligand binding site and the solvent. This allowed to build a kinetic model capable of describing the complete time course of the CO rebinding kinetics to Tf-trHb.
Highlights
The dynamics of ligand binding after photolysis in heme proteins is a complex phenomenon that entails a sequence of distinct events covering more than 10 time decades
Four spectral features are evident: two negative contributions corresponding to the Soret and Q bands bleaching (B) and two positive bands due to excited state absorption (ESA)
Excited State Dynamics Sub-nanosecond processes are known to include the formation of transient excited states with the subsequent structural response of the porphyrin macrocycle to the new electronic configuration, followed by the non-radiative relaxation to the ground state [9,35,36,37,38,39,40,41,42,43,44]
Summary
The dynamics of ligand binding after photolysis in heme proteins is a complex phenomenon that entails a sequence of distinct events covering more than 10 time decades. The possible scenarios of ligand rebinding or escape are believed to be key descriptors of the reversible ligand recognition and binding at the microscopic level and account for the observed thermodynamic and kinetic behavior at the basis of hemoglobin reactivity In this framework, laser photolysis techniques as applied in solution [12], in glasses [13] or on protein crystals [10] have contributed to the elucidation of the molecular machinery that governs ligand recognition and binding in vertebrate globins
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