Physiologically relevant reactions of myoglobin and hemoglobin with nitric oxide
Foley, Erin Williams
Olson, John S.
Doctor of Philosophy
Recent clinical trials involving administration of a cell-free hemoglobin based blood substitute have shown that several detrimental side effects are the result of reactions with nitric oxide produced by endothelial cells. We have studied key reactions between NO and hemoglobin to determine which active site amino acids influence the reaction rates, to construct mutants that have reduced rates of NO scavenging and to identify any potentially toxic reaction intermediates. A library of over 100 recombinant myoglobins and hemoglobins was used to examine: (1) reversible binding of NO to reduced Fe(II) forms, (2) reversible binding to ferric Fe(III) forms; (3) NO dioxygenation by the Fe(II)-O 2 complex, (4) oxidation of Fe(II)NO complexes in the presence of oxygen, and (5) anaerobic autoreduction of Fe(III) complexes. Results from these studies will be crucial not only for identifying problems with current blood substitutes, but also for designing new recombinant hemoglobins for the next generation of blood substitutes. The rates of reversible NO binding to ferrous and ferric myoglobin are governed largely by the free energy required to replace non-covalent and coordinated distal pocket water, respectively. Steric constraints governing the rate of ligand entry into the distal pocket play a smaller role in dictating binding rates. NO dioxygenation by oxyhemoglobin is the major cause of the hypertensive effect of extracellular hemoglobin. This reaction generates a high-spin intermediate, which quickly isomerizes to nitrate to be released from the heme pocket. Studies with H64Q mutant myoglobin suggest that a ferryl intermediate may also occur during the internal peroxynitrite isomerization reaction. Autooxidation of ferrous nitrosyl complexes is directly governed by the rate of NO dissociation. This reaction proceeds by a rapid binding of oxygen to the vacant binding site, and the displaced NO undergoes dioxygenation by the newly formed oxymyoglobin. Autoreduction of ferric nitrosyl complexes proceeds by nucleophilic addition of a hydroxide ion to the iron-bound NO, generating reduced iron and nitrous acid. The rate of autoreduction shows little dependence on distal pocket structure. In contrast, the geometry of the proximal histidine plays a key role in regulating reduction of ferric nitrosyl complexes.