Heme Reactivity is Uncoupled from Quaternary Structure in Gel-Encapsulated Hemoglobin: A Resonance Raman Spectroscopic Study

Abstract

Encapsulation of hemoglobin (Hb) in silica gel preserves structure and function, but greatly slows protein motion, thereby providing access to intermediates along the allosteric pathway that are inaccessible in solution. Resonance Raman (RR) spectroscopy with visible and ultraviolet laser excitation provides probes of heme reactivity and of key tertiary and quaternary contacts. These probes were monitored in gels after deoxygenation of oxyHb and after CO binding to deoxyHb, which intiate conformational change in the R-T and T-R directions, respectively. The spectra establish that quaternary structure change in the gel takes a week or more, but that the evolution of heme reactivity, as monitored by the Fe-histidine stretching vibration, νFeHis, is completed within two days, and is therefore uncoupled from the quaternary structure. Within each quaternary structure, the evolving νFeHis frequencies span the full range of values between those previously associated with the high- and low-affinity end states, R and T. This result supports the tertiary two-state (TTS) model, in which the Hb subunits can adopt high- and low-affinity tertiary structures, r and t, within each quaternary state. The spectra also reveal different tertiary pathways, involving the breaking and re-formation of E and F inter-helical contacts in the R-T direction but not the T-R direction. In the latter, tertiary motions are restricted by the T quaternary contacts.

Experiments on Hemoglobin in Single Crystals and Silica Gels Distinguish among Allosteric Models

“…Our calculations of oxygen affinity and populations of tertiary conformations show that only the TTS model is consistent with the experimental results. oxygen binding to hemoglobin single crystals (26,27), first carried out by Mozzarelli et al (28), and to hemoglobin encapsulated in silica gels, a method introduced by Shibayama and Saigo (29), where one quaternary structure is stable for hours to days (30,31). The single-crystal binding experiments showed that oxygen binding to the T quaternary structure is noncooperative (Fig.…”

A simple biofuel cell cathode with human red blood cells as electrocatalysts for oxygen reduction reaction

“…The four heme groups are located relatively near the surface of the protein shell the same as the family of peroxidases (Jones et al, 2012;Chen et al, 2009;Scheller et al, 2005;Perutz et al, 1968). In the Hb electrochemistry, it has been believed that the strong adsorption of proteins onto the electrode surfaces brought about a denaturation of the protein structure and slow electron transfer characteristics with the electrodes.…”

Immobilization of Proteins in Silica Gel: Biochemical and Biophysical Properties

“…Spiro and coworkers found that T quaternary structure markers do not appear in the resonance Raman spectra for about 5 days at 22 °C, which is orders of magnitude longer than the longest time of 1,000 s used in the continuous photolysis experiments [128,136]. Additionally, since the Raman experiments were carried out at complete deoxygenation, the observed R T rate is higher than the one observed at partial photolysis, as occurs in the continuous photolysis experiments.…”

“…In the case of T state encapsulated Hb allosteric effects have also been evidenced [57,58]. Monitoring the changes in the optical or resonance Raman spectra of R-state oxy-Hb gels after complete removal of oxygen demonstrated that the R T transition for the fully unliganded Hb can be slowed down by up to 10 orders of magnitude [58,80,83,92,[126][127][128]. Thus, the R T transition is not prevented by the gel matrix, rather slowed down by several orders of magnitude so that intermediate short lived Hb states, that are unstable in aqueous solutions, can be investigated with conventional spectroscopic techniques.…”