Effect of Occluded Ligand Migration on the Kinetics and Structural Dynamics of Homodimeric Hemoglobin

Abstract

Small molecules such as molecular oxygen, nitric oxide, and carbon monoxide play important roles in life, and many proteins require the transport of small molecules to and from the bulk solvent for their function. Ligand migration within a protein molecule is expected to be closely related to the overall structural changes of the protein, but the detailed and quantitative connection remains elusive. For example, despite numerous studies, how occluded ligand migration affects the kinetics and structural dynamics of the R−T transition remains unclear. To shed light on this issue, we chose homodimeric hemoglobin (HbI) with the I114F mutation (I114F), which is known to interfere with ligand migration between the primary and secondary docking sites, and studied its kinetics and structural dynamics using time-resolved X-ray solution scattering. The kinetic analysis shows that I114F has three structurally distinct intermediates (I 1 , I 2 , and I 3 ) as in the wild type (WT), but its geminate CO recombination occurs directly from I 1 without the path via I 2 observed in WT. Moreover, the structural transitions, which involve ligand migration (the transitions from I 1 to I 2 and from I 3 to the initial state), are decelerated compared to WT. The structural analysis revealed that I114F involves generally smaller structural changes in all three intermediates compared to WT.

Effect of the abolition of intersubunit salt bridges on allosteric protein structural dynamics

“…We note that the intermediates of K30D have two substrates of the fully photolyzed and partially photolyzed forms, which are structurally indistinguishable from each other. The same was revealed from previous laser power dependency studies on WT and other mutants, 35–38 which provided direct evidence that the partially photolyzed dimer subunit undergoes the same structural evolution as the fully photolyzed subunit, directly demonstrating the allosteric regulation of HbI.…”

“…As detailed in the ESI, † SVD and the global fit of left singular vectors (lSVs) show that the kinetics involves four structurally distinct intermediates and five time constants of 4.6 (±0.7) ns, 47 (±13) ns, 588 (±81) ns, 616 (±109) μs, and 5.34 (±5.22) ms. The kinetics of all the HbI proteins so far studied with TRXSS, including WT, F97Y, 37 T72V, 36 and I114F 35 can be explained with the same kinetic framework with only minor variations. This framework has three intermediates, a transition from the first intermediate to the second intermediate (I 1 → I 2 ), a biphasic transition from the second intermediate to the third intermediate (I 2 → I 3 ), and bimolecular recombination to recover CO-bound HbI(CO) 2 .…”

“…In previous studies, it was found that I WT 1 and I WT 2 have R-like structures, whereas I WT 3 has a T-like structure. 35–38 Actually, I WT 1 (RMSD R : 0.4 ± 0.05 Å and RMSD T : 0.7 ± 0.05 Å) and I WT 2 (RMSD R : 0.4 ± 0.06 Å and RMSD T : 0.7 ± 0.05 Å) have larger RMSD T than RMSD R , whereas I WT 3 (RMSD R : 0.7 ± 0.05 Å and RMSD T : 0.4 ± 0.04 Å) has a larger RMSD R compared to RMSD T , confirming that I WT 1 and I WT 2 have R-like structures and I WT 3 has a T-like structure. For K30D, I K30D 1 (RMSD R : 0.4 ± 0.05 Å and RMSD T : 0.8 ± 0.06 Å) and I K30D 2 (RMSD R : 0.5 ± 0.05 Å and RMSD T : 0.8 ± 0.06 Å) exhibit larger RMSD T than RMSD R , as in WT, indicating that I K30D 1 and I K30D 2 have R-like structures.…”

“…For the data analysis, we removed the thermal heating contribution using a well-established method. [35][36][37][38] During the experiment, the temperature was maintained at 25 C by a stream of cold nitrogen gas (Oxford Cryostream).…”

Ultrafast coherent motion and helix rearrangement of homodimeric hemoglobin visualized with femtosecond X-ray solution scattering

“…1a ) 1 – 8 . Notably, time-resolved experimental techniques such as time-resolved optical spectroscopy 9 – 12 , time-resolved X-ray crystallography (TRXC) 13 – 15 , and time-resolved X-ray solution scattering (TRXSS) 16 – 18 , which is also known as time-resolved X-ray liquidography, have been applied to HbI and its mutants to elucidate the structural transition of HbI upon ligand dissociation. Those studies revealed the structural origin of functional characteristics of HbI, such as cooperativity in ligand binding.…”