Oxygen-Binding Proteins and Hemoglobin

These flashcards cover key vocabulary related to the structure and function of oxygen-binding proteins, including myoglobin and hemoglobin, and their interactions with ligands like oxygen and carbon monoxide.

Globins

Oxygen-binding proteins that include myoglobin and hemoglobin.

Myoglobin

A globin protein that stores oxygen in muscle tissues. Myoglobin has 1 chain and cofactor

Hemoglobin

A globin protein that transports oxygen in the blood. Hemoglobin has 4 chains and 4 cofactors

Heme

An organometallic compound containing iron that binds oxygen. Porphyrin and iron.

Carbon Monoxide (CO)

A toxic gas that competes with oxygen for binding sites on hemoglobin.

Bohr Effect

The phenomenon where increased CO2 and decreased pH lead to lower affinity of hemoglobin for oxygen.

2,3-Bisphosphoglycerate (2,3-BPG)

A molecule that stabilizes the T state of hemoglobin, promoting oxygen release. It is a negative heterotropic regulator. Produced from an intermediate in glycolysis. 2,3-BPG allows for O2 release in the tissues and adaptation changes in altitude.

T state

The tense state of hemoglobin with lower affinity for oxygen. When oxygen leaves, the porphyrin “bows” and since the porphyrin is sp2 hybridizes it wants to stay flat but iron wants to be bent in. Heme is trying to buckle because missing a ligand. 

R state

The relaxed state of hemoglobin with higher affinity for oxygen. When oxygen is bound, it does not buckle.

Cooperativity

The phenomenon where the binding of one ligand increases the affinity for additional ligands.

Sickle-cell anemia

A genetic mutation in hemoglobin that causes red blood cells to adopt a sickle shape.

Biological problems and solution of globins

Problem: Protein side chains lack affinity for O2, some transition metals bind O2 well but would generate free radicals if free in solution, organometallic compounds such as heme are more suitable, but Fe2+ in free heme could be oxidized to Fe3+. Solution: capture the oxygen molecule with heme that is protein bound.

Porphyrin

A cyclic compound that forms the core structure of heme, facilitating oxygen binding in hemoglobin. Organic part

Binding of carbon monoxide

CO is highly toxic as it competes with oxygen. It blocks the function of myoglobin, hemoglobin, and mitochondrial cytochromes that are involved in oxidative phosphorylation. CO binds over 20,000 times better than O2 to heme. Protein pocket decreases affinity for CO but it still binds about 250 times better than oxygen.

Spectroscopic detection of oxygen binding to globins

The heme group is a strong chromophore that absorbs both in the ultraviolet and visible range. Ferrous form (Fe2+) without oxygen has an intense Soret band at 429 nm. Oxygen binding alters the electronic properties of the heme and shifts the position of the Soret band to 414 nm. Binding of oxygen can be monitored by UV-Vis spectrophotometry. Deoxyhemoglobin (in venous blood) appears purplish in color and oxyhemoglobin (in arterial blood) is red.

Kinetics of binding ligand to protein

The kinetics is described by the association rate constant Ka or the dissociation rate constant Kd. After some time, the process will reach equillibrium. The association constant (Ka) measures how quickly a ligand binds to a protein, while the dissociation constant (Kd) reflects how readily the ligand releases. These constants help understand the dynamics of ligand-protein interactions.

Graphical analysis of binding

Fraction of bound sites depends on the free ligand concentration and Kd. Experimentally, ligand concentration is known and Kd can be determined graphically or via least-squares regression. Small Kd binds strong. “How much must we add to get X amount bound.” Is analyzed using a binding curve where the x-axis represents ligand concentration and the y-axis shows the fraction of occupied binding sites.

Oxygen binding to myoglobin

Myoglobin is a muscle protein that binds oxygen, facilitating oxygen storage and transport within muscle cells. It has a higher affinity for oxygen than hemoglobin, allowing for effective oxygen delivery during periods of high metabolic demand. Partial pressure of O2 on the x axis and percent bound on the y axis.

Cooperativity

Cooperativity refers to the phenomenon where the binding of one ligand to a protein affects the binding of subsequent ligands. In hemoglobin, this results in an increased affinity for oxygen after the initial binding, enabling more efficient oxygen transport. Results in conformational change.

The Hill plot of cooperativity

Straight line indicates that protein only has 1 binding site (myoglobin has 1 binding site, no cooperativity). Curved line indicates protein has more than 2 binding sites. Hill coefficient for hemoglobin is 3.

oxy state of hemoglobin

fewer interactions, more flexible, higher affinity for O2 (red)

deoxy state of hemoglobin

more interactions, more stable, lower affinity for O2 (blue)

T state of hemoglobin

deoxy state (blue)

R state of hemoglobin

oxy state (red)

R and T state of hemoglobin

O2 binding triggers a T to R conformation change, breaking ion pairs between the alpha1 and beta2 interface.

pH effect on O2 binding to hemoglobin

actively metabolizing tissues generate H+ lowering the pH of the blood near the tissues relative to the lungs (catalyzed by carbonic anhydrase). Hb affinity for oxygen depends on the pH, as H+ binds to Hb and stabilizes the T state. The pH difference between lungs and metabolic tissues increases the efficiency of the O2 transport. Known as the Bohr effect.

Fetal hemoglobin

Fetal hemoglobin has a higher affinity for O2. It binds differently than maternal and fetal hemoglobin pulls oxygen away from maternal hemoglobin.

Hemoglobin and CO2 export

CO2 is produced by metabolism in tissues and must be exported. 15-20% is exported in the form of a carbamate on the amino terminal residues of each of the polypeptide subunits. The formation of a carbamate yields a protein that can contribute to the Bohr effect. Carbamate forms additional salt bridges, stabilizing the T-state. The rest of the CO2 is exported as dissolved bicarbonate.