Thermodynamics and spectroscopy of hemoglobin and myoglobin
Hemoglobin: a cooperative protein
Hemoglobin and myoglobin are both oxygen transport and storage proteins. The distinction is generally that hemoglobin is found in vascular tissue or extracellular fluids where it carries diatomic oxygen from the lungs or other capture organs to the tissues. Many hemoglobins are multimeric consisting of two or four subunits. Each subunit has a structure similar to myoglobin, containing one heme capable of binding diatomic oxygen. The subunits of hemoglobins are capable of communicating such that the binding of oxygen causes structural changes that affect the binding of oxygen in other subunits. For example, the binding of the first oxygen molecule to the first heme causes a change in structure that increases the oxygen binding affinity in the other subunits. Similarly the dissociation of oxygen causes changes in structure that reduce the affinity of oxygen in other subunits and results in cooperative unloading of oxygen in tissues.
We begin by focusing on the properties of myoglobin. Myoglobin is located in muscle and its an oxygen storage protein. Transport of diatomic oxygen may also occur by passive diffusion between various myoglobin proteins. The study of myoglobin has used many spectroscopic and thermodynamic methods to study both binding rate constants and affinity. In addition, numerous structures have given a model for stabilization of oxygen due to hydrogen bonding by a distal histidine. In this way, myoglobin discriminates against the binding of carbon monoxide (CO). CO is an endogenous poison produced by the degradation of heme by heme oxygenase. Myoglobin, hemoglobin and other heme proteins must favor the binding of oxygen over CO despite the fact that instrinsic affinity fo CO is 20,000 times higher than O2 in free heme.
Kinetic models for geminate and bimolecular recombination
Studies of carbon monoxide (CO) rebinding
CO recombination is studied extensively as a model for O2 recombination. Both CO and O2 can be photolyzed by a short laser pulse. However, one third of the O2 apparently does not dissociate, one third of the O2 recombines rapidly within picoseconds and one third recomgines on the nanosection time scale. CO by contrast has nanosecond (geminate) recomination and some CO can escape from the protein.. Moreover, diatomic oxygen causes autooxidation Mb, which results in formation of ferric heme with a half life of 45 minutes. Thus, it is difficult to conduct a kinetic experiment because the sample is autooxidized as the experiment proceeds. CO on the other hand forms a stable complex and can be photolyzed thousands or tens of thousands of times reproducibly without autooxidation or other side reactions. Although CO is not the physiological ligand it is clearly the ligand of choice for laboratory studies of the kinetics of ligand rebinding.
The kinetics of CO rebinding can be divided into geminate and bimolecular proceses. A geminate process occurs within the distal pocket on the nanosecond time scale. It is an immediate recombination. The reason that it is "slow", e.g. compared to O2 rebinding is that the Fe atom changes from low spin when CO is bound to high spin when CO is dissociated. Thus, CO recomination must overcome the barrier of a change of spin state from S=2 to S=0. Therefore, a competing process is escape of CO into the solvent. Once this occurs CO rebinding is a bimolecular process. The CO that recombines is not necessarily the same one that escaped. CO rebinding depends on the CO concentration in solution. Therefore, the bimolecular process has a pseudo-first-order rate constant. The kinetic scheme is explained in the link below.
