ABSTRACT
Many biochemical reactions are complex and involve consecutive equilibria along the reaction pathway (1). Further complicating matters, the rate of passage between neighboring free energy minima can be modulated by conformational changes of an enzyme. To understand the mechanism of reaction in such complex systems, it is crucial to probe, in detail, intermediates along the reaction pathway. Myoglobin (Mb) is an 18 kDa heme protein that has long served as a model system for probing protein control of ligand binding and discrimination (2). The active binding site in Mb consists of an iron(II)-containing porphyrin known as a heme that is embedded within the hydrophobic interior of the globular protein (see Fig. 1). This heme reversibly binds O2 as well as biologically relevant [and infrared (IR) active] NO (3) and CO (4). According to sequence homologies among 60 mammalian species (5), most residues in the immediate vicinity of the distal
side of the heme are 100% conserved. These highly conserved residues evidently mediate the passage of ligands to and from the active binding site and modulate their binding affinities. For example, CO binds to free heme about 103 –104 times as strongly as O2 (6) but binds to the heme in Mb only 30 times as strongly as O2 (7). Because CO is produced endogenously by the metabolism of heme (8), discrimination against this toxic ligand is thought to be biologically important. The protein structure also influences the kinetics of geminate rebinding, with some rates being time-dependent and slaved corresponds to conformational changes of the protein.
