8 Oxygen Transport

monomeric helical protein that contains heme & delivers oxygen in muscle

a nonprotein group forming part of or combined with a protein

• heme in hemoglobin; can be found in a deep, non-polar pocket to sequester it from water (helps keep the Fe in the +2 state)

• when exposed to water, Fe is oxidized to the 3+ state where it can't hold oxygen

• if sequestered in a nonpolar region of the protein, heme can maintain its oxygen-carrying Fe²⁺ state

1. both proteins have high alpha-helical content

2. contain a heme prosthetic group, which is the oxygen carrier

oxygenation → Hb Fe²⁺ & O₂ (oxymyoglobin)

oxidation → Hb: Fe³⁺ (metmyoglobin)

oxygenated heme is bright red

oxiDIZed heme is brownish

deoxygenated heme is more blue

• in isolated heme, CO binds to Fe at a 180° angle & O2 binds at 120° (bent)



• however in myoglobin histidine E7 forces a substrate to bind at 120°, therefore optimal CO binding is prevented, but O₂ binding is ideal



* CO binds more weakly to heme in myoglobin than to free heme allowing oxygen binding in myoglobin*

• when it binds to oxygen, the oxygenated ferrous ion (Fe²⁺) moves into the plane of the porphyrin ring (straightens it out)

• • this pulls along some of the protein with it (mediated by histidine F8)
• - movement of F8 helps the protein sense whether or not O₂ is bound to the heme group

• myoglobin is a monomer; the change in structure upon oxygenation has little consequence

only a minor conformational change

• one forms a ligand to the iron that moves upon oxygenation

• the other reduces CO binding by requiring binding to occur at a 120° angle

• transports oxygen between the lungs & tissues

• is made up of 4 myoglobin-like subunits: 2 alpha & 2 beta subunits (a dimer of alpha/beta units)

• it’s globular

• has 4 hemes in total, 1 in each subunit

each alpha subunit is in close contact with both beta subunits

• in contrast, there are FEW interactions between like subunits

Just know they're nearly identical

hyperbolic

suggests that simple equilibrium processes are involved in binding & release

Sigmoidal (sort of looks like an S)

indicates cooperativity among multiple binding sites (binding of oxygen to 1 site influences the oxygen affinity of the remaining sites on HbA)

represents the level of dissolved oxygen in plasma, or whatever surroundings/environment being discussed

describes the effect ligands bound to a macromolecule have on binding a 2nd or additional ligand to that same macromolecule

• • n > 1 = positive cooperatively
• - binding of 1 ligand promotes the binding of another

• n = 1: no cooperativity

• • n < 1 negative cooperatively
• - binding 1 ligand inhibits binding of a second

M = 1 (binding of 1 ligand has no effect on the binding of another)

Hb = 2.8 (binding of 1 ligand promotes the binding of another)

conformational state of DEoxyhemoglobin

• in the absence of oxygen, the T form is more stable than the R form because it contains more electrostatic & hydrogen bonds

• *has a greater affinity for protons

conformational state of OXYhemoglobin

• R state has the greatest affinity for oxygen

• initial trigger is the movement of the Fe2+ ion upon oxygenation

• leads to a conformational change in a subunit

• the alpha subunit is in contact with the beta subunit (& vice versa), causing the other subunits to take on the oxygen-binding conformation

they differ in the ARRANGEMENT of the subunits although the subunits themselves BARELY change structure

the oxygen binding affinity of hemoglobin is reduced by increased CO₂ & H⁺ concentration (low pH)

• this allows hemoglobin to deliver more oxygen to actively metabolizing tissues

CO₂ & low pH promote the release of oxygen by lowering the oxygen affinity of hemoglobin

regulation of a protein by binding an effector molecule at a site other than the enzyme's active site

eg. H+ & CO2 are negative allosteric effectors

Carbon dioxide binds to the N-terminal amino groups of the alpha subunits

• this is DIFFERENT from carbon monoxide, which binds to the oxygen binding site

• CO2 reacts with an N-terminal amino group of an alpha subunit & forms a Carbamate

• Carbamate stabilizes the Taut (deoxy) state of hemoglobin, lowering its oxygen affinity

another molecule that lowers hemoglobin's oxygen affinity by binding & stabilizing deoxyhemoglobin (Taut)

• BPG is negatively charged & binds in a positively charged cavity formed by the beta chains of hemoglobin

• Fetal (HbF) is composed of 2 alpha & 2 gamma subunits

• Adult (HbA) is composed of 2 alpha & 2 beta subunits

FETAL

• the gamma subunits in HbF have fewer positively charged amino acid residues in the BPG binding region than beta subunits in HbA

• HbF binds oxygen with a higher affinity because BPG binds less to fetal hemoglobin

• • 1st described genetic disease
• • characterized by sickled appearance of RBCs due to insoluble HbS (hemoglobin)
• • result = clogged capillaries & general organ damage causing pain (+ anemia, jaundice, frequent infections)
• • individuals can either be homo (more severely affected) or heterozygous (range of severity)
• • 1/12 african americans = heteros
• • 1/100 hispanic americans = heteros
• • 80,000 with disease in US

point mutation converting Glutamate → Valine on the exterior of the beta subunit

• results in an altered charge of the beta subunit

• Glutamate is very negatively charged; Valine is neutral; electrophoresis of the Hb can be used to diagnose (HbS won’t migrate as much as HbA toward the + Anode because it’s less negative)

• only then are the beta subunits of adult hemoglobin produced

• symptoms associated with sickle cell anemia correlate with decreased synthesis of gamma subunits & increased synthesis of beta subunits

• a hydrophobic hole is formed in both HbA & HbS in the deoxy (taut) form

• a hydrophobic knob is present on the beta subunit of HbS in both oxy & deoxy forms (R & T)



• in areas of low oxygen (eg. high altitude), HbS polymerizes (knobs fit into holes) & distorts red cells, leading to clogging of capillaries & blood vessels

• long term result = organ damage

• Antibiotics: prevents 2ndary infections

• Hydroxyurea: stimulates production of HbF

• • Bone marrow transplantation: replaces HbS with HbA
• - cure, but have to worry about immunological consequences

• Gene therapy: works in mice

Fe³⁺ (ferric)

this is an abnormal state that can't bind oxygen

• shortness of breath
• cyanosis
• mental status changes (~50%)

• diagnosed by the absorption spectrum of the blood

1. ingestion of large amounts of oxidizing agents such as nitrates (eg. fertilizer found in well water)

2. defects can also arise from the lack of maintenance of reducing agent in RBCs

3. mutations in the heme-binding pocket of Hb can promote HbM (mutation of Histidine F8 → Tyrosine)

by administering reducing agents like vitamin C or methylene blue

• • an imbalance of the synthesis of the alpha & beta subunits of hemoglobin
• • ~1000 cases in the US of severe disease
• • alpha thalessemias have defective levels of alpha subunits; beta, the beta subunits
• • treatment usually involved blood transfusion