Distal and proximal control of oxygen binding and release in hemeproteins

Abstract

Flash photolysis studies at cryogenic temperatures have been used to investigate the intermediates of ligand binding in myoglobin (Mb) and hemoglobin (Hb). Low temperature photoproduct yields provide a probe of the inner barrier to ligand binding, i.e. the reaction of the ligand with the heme iron. We find that the 10 K photoproduct yield of carbonmonoxy myoglobin (MbCO) (1.0) is greater than oxymyoglobin (MbO{dollar}\sb2{dollar}) (0.50 {dollar}\pm{dollar} 0.05), implying a higher inner barrier for CO binding to myoglobin. The photoproduct yield of cobalt-substituted oxymyoglobin (CoMbO{dollar}\sb2{dollar}) (0.55 {dollar}\pm{dollar} 0.05) is similar to MbO{dollar}\sb2{dollar}, indicating that substitution of cobalt for iron does not affect the inner barrier to oxygen binding. At low pH, the 10 K photoproduct yields for MbO{dollar}\sb2{dollar} (pH 5: 0.18 A) and CoMbO{dollar}\sb2{dollar} (pH 4: 0.20 A) both decrease; however, the pK's for the photoproduct yield versus pH titration curves are different for MbO{dollar}\sb2{dollar} (pK = 5.7) and CoMbO{dollar}\sb2{dollar} (pK = 4.8). This pH dependence is centered on the titration of the distal histidine; the photoproduct yield of the mutant, Mb(H64Q)O{dollar}\sb2{dollar}, where the hydrogen bond to the distal glutamine does not titrate with pH, remains constant (0.50 {dollar}\pm{dollar} 0.05) from pH 4.8 to pH 9.;The decrease in photoproduct yield for MbO{dollar}\sb2{dollar} at low pH implies distal influences on the inner barrier to oxygen binding. We have used the CO stretching frequency of horse skeletal muscle myoglobin to probe distal pocket structure as a function of pH in the ligand-bound (A states) and photoproduct states (B states). The pH-dependence of the A{dollar}\sb1{dollar} state fraction in horse MbCO follows the same pH dependence as the photoproduct yield for horse MbO{dollar}\sb2{dollar}, suggesting functionally important conformational substates in MbO{dollar}\sb2{dollar}, where the population of the low-pH substate lowers the inner barrier to oxygen binding.;Photolyzed/unphotolyzed difference infrared spectroscopy has been used to identify a single photolyzable {dollar}\nu{dollar}(O-O), which falls at 1131 cm{dollar}\sp{lcub}-1{rcub}{dollar} for MbO{dollar}\sb2{dollar} and 1138 cm{dollar}\sp{lcub}-1{rcub}{dollar} for CoMbO{dollar}\sb2{dollar}. Since previous work, using infrared difference spectra of isotopically labelled MbO{dollar}\sb2{dollar}, has identified two dioxygen stretching frequencies (Potter, et al., (1987) Biochemistry, 26, 4669), we suggest that the photolyzable fraction of MbO{dollar}\sb2{dollar} molecules corresponds to a hydrogen-bonded conformational substate of MbO{dollar}\sb2{dollar} (1131 cm{dollar}\sp{lcub}-1{rcub}{dollar}) and the unphotolyzable fraction corresponds to a non-hydrogen bonded conformational substate of MbO{dollar}\sb2{dollar} ({dollar}\sim{dollar}1150 cm{dollar}\sp{lcub}-1{rcub}{dollar}).;A study of distal control of the inner barrier has been extending to the R and T states of carp HbCO. Through infrared spectroscopy, we find a higher photoproduct yield for the T state (1.0) than the R state (0.80 {dollar}\pm{dollar} 0.05), implying a higher barrier to CO binding in the T state. In the T state, a single conformational substate (A,) exists with respect to the CO stretching frequency. The T {dollar}\to{dollar} R state transition results in population the A{dollar}\sb0{dollar} conformation in the R state, which rebinds CO faster, suggesting distal pocket involvement in the mechanism of hemoglobin cooperativity.