Direct observation of photolysis-induced tertiary structural changes in hemoglobin

Abstract

Human Hb, an ␣2␤2 tetrameric oxygen transport protein that switches from a T (tense) to an R (relaxed) quaternary structure during oxygenation, has long served as a model for studying protein allostery in general. Time-resolved spectroscopic measurements after photodissociation of CO-liganded Hb have played a central role in exploring both protein dynamical responses and molecular cooperativity, but the direct visualization and the structural consequences of photodeligation have not yet been reported. Here we present an x-ray study of structural changes induced by photodissociation of half-liganded T-state and fully liganded Rstate human Hb at cryogenic temperatures (25-35 K). On photodissociation of CO, structural changes involving the heme and the F-helix are more marked in the ␣ subunit than in the ␤ subunit, and more subtle in the R state than in the T state. Photodeligation causes a significant sliding motion of the T-state ␤ heme. Our results establish that the structural basis of the low affinity of the T state is radically different between the subunits, because of differences in the packing and chemical tension at the hemes.A llosteric transitions allow rapid regulation of protein function in biological systems. There is a wealth of structural data regarding the end states of allosteric transitions of various proteins (1), but very little is known about how they work. A typical example of allosteric regulation is the cooperative oxygen binding by human Hb. The Hb molecule is a heterotetramer consisting of two ␣ subunits and two ␤ subunits, ␣ 2 ␤ 2 , which are arranged as a dimer of ␣␤ dimers. Each subunit contains one heme group to which one oxygen molecule binds reversibly. The oxygen affinity of each subunit rises as the other hemes in the same tetramer become saturated with oxygen. The binding of oxygen by Hb is therefore cooperative, allowing efficient transport of oxygen in the blood. Crystal structures of fully unliganded tense (T)-state and fully liganded relaxed (R)-state Hb reveal multiple differences at both tertiary and quaternary levels (2-5), but, as is the case for most other allosteric proteins, through what mechanism these different structures impact ligand reactivity is still not well understood (for review, see ref. 6). An essential part of this question is understanding the mechanism of restraints on ligand binding in the T state, because the oxygen affinity of the R state is close to that of isolated ␣ and ␤ subunits. In the early 1970s, Perutz suggested that the low affinity of the T state is caused by tension in the iron-proximal His(F8) bond in the liganded T state (7), but several lines of evidence show that the ligand affinity of the ␣ and ␤ subunits is regulated by different mechanisms (8)(9)(10)(11)(12). Despite the wealth of data from x-ray crystallography, a means of defining the stereochemical basis unambiguously for the difference in oxygen affinity between the T and R states, which is only a few kcal͞mol, has not been determined (13). It is important to note that the larg...

A quantum-chemical picture of hemoglobin affinity

“…In addition, strain is focused in the FG corner of the ␣-chains, at the site of the important hinge quaternary contact, which is known to be critical for T state stabilization and cooperative ligand binding. These results are strongly supported by the cryogenic crystallography of Shibayama and coworkers (23). They showed that photodissociation of CO in the T structure (produced in Fe,Ni hybrids) leads to significant movement of the ␣-chain F-helix, including the proximal histidine that it contains, which is propagated to the FG corner.…”

“…These were calculated by in silico treatment (see Methods) of the highest-resolution crystal structures available for the two forms of Hb (21). To dissociate HbCO, we stretched the Fe-C bond until the CO dissociated and adopted a position adjacent to and parallel to the heme, the geometry observed in photodissociation experiments of myoglobin (Mb) (22) and Hb (23,24). In deoxyHb a CO molecule was allowed to diffuse into the heme pocket and then bind to the heme.…”

Allosteric action in real time: Time-resolved crystallographic studies of a cooperative dimeric hemoglobin

“…Results to date suggest a multistep pathway, with key tertiary structural transitions taking place within a few microseconds and quaternary rearrangements occurring in the range of tens of microseconds. In addition to providing structural information on high-affinity R and low-affinity T quaternary forms at the atomic level (16)(17)(18), crystallographic experiments have also succeeded in illuminating ligand-linked structural tertiary transitions within the constraints of R or T forms (19).…”

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.…”

“…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.…”