Abstract
We report the ligand dependence of the conformer distribution in the distal heme pocket of Ascaris suum hemoglobin (Hb) studied by resonance Raman spectroscopy. The heme-bound CO is used as a spectroscopic antenna to probe the original distribution of conformers in the dioxygen derivative of Ascaris Hb, by utilizing sol-gel encapsulation. The first step is to encapsulate the dioxygen derivative in the porous sol-gel and let the gel age, thus trapping the equilibrium conformational distribution of Ascaris dioxygen Hb. In the second step, the dioxygen ligand is replaced by CO. The sol-gel environment impedes any large scale movements, drastically slowing down the conformational relaxation triggered by the ligation change, essentially "locking in" the initial quaternary and even tertiary structure of the protein. Studying the Fe-CO frequencies of the latter sample allows evaluation of the distribution of the distal heme pocket conformers that was originally associated with the dioxygen derivative. Extending the study to the Ascaris mutants allows for examination of the effect of specific residues in the distal pocket on the conformational distribution. The choice of mutants was largely based on the anticipated variation in hydrogen bonding patterns. The results show that the sol-gel encapsulation can slow or prevent re-equilibration within the distal heme pocket of Ascaris Hb and that the distribution of distal heme pocket conformers for the CO derivative of Ascaris Hb in the sol-gel is highly dependent on the history of the sample. Additionally, we report a detailed study of the CO complex of the mutants in solution for assignment of the various heme pocket conformers, and we present a comparison of the sol-gel data with solution data. The results support a picture in which the dioxygen derivative biases the population strongly toward a tightly packed configuration that favors the network of strong hydrogen bonding interactions, and suggest that Ascaris Hb is uniquely designed for dioxygen capture.
Highlights
We report the ligand dependence of the conformer distribution in the distal heme pocket of Ascaris suum hemoglobin (Hb) studied by resonance Raman spectroscopy
The results show that the sol-gel encapsulation can slow or prevent re-equilibration within the distal heme pocket of Ascaris Hb and that the distribution of distal heme pocket conformers for the CO derivative of Ascaris Hb in the sol-gel is highly dependent on the history of the sample
The results support a picture in which the dioxygen derivative biases the population strongly toward a tightly packed configuration that favors the network of strong hydrogen bonding interactions, and suggest that Ascaris Hb is uniquely designed for dioxygen capture
Summary
We report the ligand dependence of the conformer distribution in the distal heme pocket of Ascaris suum hemoglobin (Hb) studied by resonance Raman spectroscopy. It was proposed that the population giving rise to the 543/1909 –1912 cmϪ1 set of peaks corresponds to a conformation of the distal heme pocket in which the B10 and E7 residues are in close proximity to the bound CO This postulate was supported by studies of the Tyr-B10 3 Phe mutant in which the 543/1909 –1912 cmϪ1 bands were absent [9, 10]. On the basis of earlier experimental and theoretical studies (9 –11) on the determinants of the heme-bound CO stretching frequency ( see Ref. for Mycobacterium tuberculosis Hb and Ref. for a review on carbonmonoxy myoglobin), it can be concluded that strong polar interactions, such as the ones arising from the close proximity of the Tyr-B10 phenol proton to the bound CO, are the dominant factors that cause such high Fe–CO and low C–O frequencies The vibrational data indicate that in solution there are accessible conformations of the distal pocket of Ascaris HbCO that can support strong interactions between polar residues and the CO ligand [10], anticipated to be similar to the hydrogen bonding patterns for the oxy complex of Ascaris Hb as suggested by the kinetics (1–3, 6 –7, 9) and the crystal structures [5]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
