Differential control of heme reactivity in alpha and beta subunits of hemoglobin: a combined Raman spectroscopic and computational study.

ABSTRACT: The use of hybrid hemoglobin (Hb), with mesoheme substituted for protoheme, allows separate monitoring of the ? or ? hemes along the allosteric pathway. Using resonance Raman (rR) spectroscopy in silica gel, which greatly slows protein motions, we have observed that the Fe-histidine stretching frequency, ?FeHis, which is a monitor of heme reactivity, evolves between frequencies characteristic of the R and T states, for both ? or ? chains, prior to the quaternary R-T and T-R shifts. Computation of ?FeHis, using QM/MM and the conformational search program PELE, produced remarkable agreement with experiment. Analysis of the PELE structures showed that the ?FeHis shifts resulted from heme distortion and, in the ? chain, Fe-His bond tilting. These results support the tertiary two-state model of ligand binding (Henry et al., Biophys. Chem. 2002, 98, 149). Experimentally, the ?FeHis evolution is faster for ? than for ? chains, and pump-probe rR spectroscopy in solution reveals an inflection in the ?FeHis time course at 3 ?s for ? but not for ? hemes, an interval previously shown to be the first step in the R-T transition. In the ? chain ?FeHis dropped sharply at 20 ?s, the final step in the R-T transition. The time courses are fully consistent with recent computational mapping of the R-T transition via conjugate peak refinement by Karplus and co-workers (Fischer et al., Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 5608). The effector molecule IHP was found to lower ?FeHis selectively for ? chains within the R state, and a binding site in the ?1?2 cleft is suggested.

SUBMITTER: Jones EM 

PROVIDER: S-EPMC4353013 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature