Oxygen binding to hemoglobin and myoglobin is one of the most physiologically important reactions in human cells. Loss of oxygen and insufficient delivery has a myriad of severe health consequences, including stroke, cardiovascular dysfunction, and death of brain cells. To a large degree, the cessation of oxidative phosphorylation is the most critical physiological consequence of oxygen deprivation. However, oxygen is also a crucial substrate in other important reactions of metabolism, including the action of heme oxidases and oxygenases. Hemoglobin and myoglobin are paradigms for the study of oxygen metabolism, in that their structures and dynamics have been so extensively investigated. Clearly, the thermodynamics and kinetics of oxygen binding are well established. We contend that the nature and structure of the intermediates of 02 rebinding for globins are poorly understood. A full understanding of the control of reactivity must include details of intermediate states. Substantial progress has been made in investigating the intermediates of CO binding, and it has always been assumed that CO would provide, at least, an adequate model for 02 binding. A number of experiments, detailed in the proposal, clearly show that 02 has both different intermediates of ligand rebinding and ligand bound states. A S = 1 state for iron is probable for 02 binding. This explains the 0.2 A iron displacement for MbO2 and the presence of high proportions of S = 1 states subsequent to photolysis, and may explain why substantial fractions of 02 populations rebind quickly. We will trap intermediate states by the use of cryogenic techniques and we will examine ligand bound states at a range of temperatures. We propose the use of techniques to probe distal hemepocket, proximal hemepocket, and iron structure for MbO2 and HbO2 in different quaternary states. Ligand vibrational spectra probe the iron-oxygen bond and its distal environment. We will use FTIR spectroscopy to investigate the kinetics of ligand bound conformations. EXAFS studies can measure the iron-pyrrole nitrogen, iron-oxygen, and iron-histidine bond lengths accurately in protein compounds of defined reactivity or geminate states of photolysis. X-ray edge measurements (XANES) can measure iron displacement, charge density on the central metal, and 3d orbital occupancy. Near-IR studies of band Ill probe the proximal hemepocket configuration of the """"""""deoxy like"""""""" photoproduct intermediate states. We will also examine myoglobin mutants, where the distal histidine is replaced with other selected residues. This will probe the important hydrogen bond stabilization of 02 adducts. As a result of these investigations, we will identify the heme and hemepocket parameters that control hemoglobin cooperativity, the reactivity of conformational substates, and explain the differential reactivity of CO and 02.
| Chance, M R (1993) Low-temperature optical spectroscopy: metalloprotein structure and dynamics. Methods Enzymol 226:97-118 |
