Following Ligand Migration Pathways from Picoseconds to Milliseconds in Type II Truncated Hemoglobin from Thermobifida fusca

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.

Structural flexibility of the heme cavity in the cold‐adapted truncated hemoglobin from the Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125

“…In terms of ligand accessibility to the active site, the E7 pathway located between helices E and F was already observed in related TrHbIIs, e.g. Tf-2/2HbO and Mt-2/2HbO, suggesting similar k on values (10 4 -10 5 M À1 Ás À1 ) for the predominant CO-rebinding process in these proteins [14,15,59,60]. Nevertheless, the crystallographic and MD analyses presented in this work do not provide an explanation for the biphasic nature of CO-rebinding kinetics of Ph-2/2HbO [14,15].…”

2D‐IR spectroscopy: an additional dimension to investigate ultrafast structural dynamics

“…There is not a single technique capable of providing both dynamics and structure over such a broad time scale. It is necessary to combine different techniques to cover such extended temporal interval . Optical techniques with short laser pulses provide a unique tool to investigate the elementary processes occurring in molecular systems, thus opening the possibility to disentangle complex dynamics and helping in correlating it to the structure and functionality on short time scales from few tens of fs and up to few ns.…”

“…It is necessary to combine different techniques to cover such extended temporal interval. [2,3] Optical techniques with short laser pulses provide a unique tool to investigate the elementary processes occurring in molecular systems, thus opening the possibility to disentangle complex dynamics and helping in correlating it to the structure and functionality on short time scales from few tens of fs and up to few ns. The basic principle ruling these techniques is that excitation pulses, interacting with a molecular system at any level (electronic, vibrational etc..), put it 'instantaneously' out of equilibrium and trigger a cascade of relaxation processes eventually bringing the system back to a new equilibrium position.…”

Molecular dynamics simulations indicate that tyrosineB10 limits motions of distal histidine to regulate CO binding in soybean leghemoglobin

“…Despite a large body of research on these “new” globins worldwide, conclusive functional understanding is still lacking. Mechanistic understanding of ligand binding regulation, ligand migration, dynamics of heme pockets, role of individual amino acids in ligand stabilization and synergy between distal pocket residues in the new globins is underway and incomplete . The surge in new information has also forced a re‐assessment of the “classical” Hbs and it is imperative that complete understanding of classical systems other than the paradigms myoglobin (Mb) and Hb are necessary for better evaluation of the newer globins …”

“…Mechanistic understanding of ligand binding regulation, ligand migration, dynamics of heme pockets, role of individual amino acids in ligand stabilization and synergy between distal pocket residues in the new globins is underway and incomplete. [8][9][10][11][12][13] The surge in new information has also forced a re-assessment of the "classical" Hbs and it is imperative that complete understanding of classical systems other than the paradigms myoglobin (Mb) and Hb are necessary for better evaluation of the newer globins. 14 An enigmatic classical Hb system that demands further investigation is the plant "symbiotic" globin, leghemoglobin (Lb).…”