Real-time tracking of CO migration and binding in the α and β subunits of human hemoglobin via 150-ps time-resolved Laue crystallography

Abstract

We have developed the method of picosecond Laue crystallography and used this capability to probe ligand dynamics in tetrameric R-state hemoglobin (Hb). Time-resolved, 2 Å-resolution electron density maps of photolyzed HbCO reveal the time-dependent population of CO in the binding (A) and primary docking (B) sites of both α and β subunits from 100 ps to 10 μs. The proximity of the B site in the β subunit is about 0.25 Å closer to its A binding site, and its kBA rebinding rate (~300 μs−1) is six times faster, suggesting distal control of the rebinding dynamics. Geminate rebinding in the β subunit exhibits both prompt and delayed geminate phases. We developed a microscopic model to quantitatively explain the observed kinetics, with three states for the α subunit and four states for the β subunit. This model provides a consistent framework for interpreting rebinding kinetics reported in prior studies of both HbCO and HbO2.

Quaternary-Linked Changes in Structure and Dynamics That Modulate O2 Migration within Hemoglobin’s Gas Diffusion Tunnels

“…33 The faster phase likely corresponds to recombination from the β-distal heme site and the slower phase probably involves recombination of ligands that had diffused to the β-tunnel (Figure 3b,d,f 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 migration from Mb's distal heme site to its interior tunnel network has been established by numerous cryogenic and time-resolved X-ray crystallographic studies 17,36-42 as well as MD simulations. 13,14,[43][44][45] Both experiment and simulation support ligand diffusion between the distal heme sites and the interior cavities of HbA and Mb.…”

Sub-100-ps structural dynamics of horse heart myoglobin probed by time-resolved X-ray solution scattering

“…Although the optical spectroscopic tools have been quite successful in identifying the time scales for the formation of intermediates involved in protein transitions, their signals are not directly related to three-dimensional structure of the protein. Alternatively, time-resolved Laue crystallography can provide a combination of high time resolution and structural sensitivity [46–51], but it requires the preparation of highly-ordered and radiation–resistant single crystals, limiting its applicability to only a few model systems. More importantly, the protein motions in crystalline sample might be different from those in physiological aqueous environment where proteins actually perform their functions [48–50].…”

Size and Shape Controlled Crystallization of Hemoglobin for Advanced Crystallography

“…Adachi et al [7] and Schotte et al [8] have independently conducted X-ray analysis of the photoproduct of R-state COHb. These groups reported a 2.5 Å-resolution cryo-trapped photoproduct structure at 35 K, and time-resolved 2.0 Å-resolution electron density maps of the crystal at 15 • C, respectively.…”

“…These groups reported a 2.5 Å-resolution cryo-trapped photoproduct structure at 35 K, and time-resolved 2.0 Å-resolution electron density maps of the crystal at 15 • C, respectively. In both studies, the bipyramidal-shaped crystals of COHbA were used (with size of 30 µm in the former study [7] and~250 µm in the latter [8]). The yields of photoproducts at 35 K and 15 • C were about 50% and 15%, respectively.…”

“…However, this is not always the case, since protein crystallography experiments are becoming more diverse and challenging, and thereby requirements for crystals are also becoming more demanding in terms of size and shape. The purpose of this article is to illustrate our approach to the growth of Hb crystals to dimensions that meet the needs of advanced crystallography, including XRC combined with photoexcitation (e.g., photolysis of CO bound to Hb [7,8]) and/or spectrophotometry [9,10], neutron crystallography (NC) [11,12], and recently developed X-ray fluorescent holography (XFH) [13]. The first one requires optically thin but well-diffracting Hb crystals, while the latter two, especially XFH, require large-volume, well-ordered Hb crystals, due to feeble signals.…”