Near-infrared absorption studies of conformational relaxation and binding kinetics in hemeproteins

Abstract

The near-infrared absorption band at about 760 nm (Band III) in hemeproteins is sensitive to the local heme conformation of equilibrium and nonequilibrium five coordinate ferrous high spin hemes. It is absent in six coordinate liganded forms. The time evolution of this absorption band subsequent to photodissociation of liganded hemoglobin or myoglobin provides detailed information regarding both conformational relaxation from nonequilibrium to equilibrium states and the thermally driven fluctuations that result in the transition from inhomogeneous to homogeneous ligand rebinding kinetic.;In this thesis, the time dependent properties of Band III are used to study the dynamics of hemeproteins. Results are presented from three studies on hemoglobin. The first deals with conformational averaging due to thermal fluctuations. The second reveals relaxation properties in mutant human hemoglobin, hemoglobin Ypsilanti. In the third study, a homo-dimeric clam hemoglobin (Scapharca inaequivalvis) shows high cooperativity in ligand binding. This is the smallest hemoglobin that exhibits cooperativity.;Three temperature and/or viscosity dependent phenomena are observed for the 75% glycerol sample ({dollar}\sim{dollar}30 centerpoise) of the photoproducts of carboxy adult human hemoglobin: kinetic hole burning, subsequent dynamic hole filling and additional broadening of band III. At high viscosity ({dollar}\geq{dollar}400 cp) conformational relaxation slows dramatically and both kinetic hole burning followed by the filling in of the "hole" are observed. As the temperature is lowered conformational relaxation slows and finally ceases.;Hemoglobin Ypsilanti ({dollar}\beta{dollar}99Asp {dollar}\to{dollar} Tyr) has high affinity for ligand binding and is not cooperative. The experiments show that unlike HbA there is little structural relaxation occurring after photodissociation of its CO derivatives and the photoproduct appears to be locked into an R-like conformation.;Scapharca inaequivalvis clam hemoglobin (HbI) is a homodimer with two ligand binding sites. It is highly cooperative with a Hill coefficient, n = 1.5 despite its rigidity in structure. Structural relaxation after photolysis of its CO derivative is fast and monophasic compared to tetrameric hemoglobins. It is fully relaxed within 100 ns while in tetrameric hemoglobins the relaxations extend out to 20 {dollar}\mu{dollar}s or longer.