Ch. 7 Protein Function and Evolution

Myoglobin and Hemoglobin

• Both are essential for oxygen need• Myoglobin stores O2 in the muscle

• Hemoglobin transports O2 to tissues and CO2

and H+ back to lungs• The 2o structure of hemoglobin resembles

myoglobin but the 4o structure allows for interactions that are central to its function.

Structure

• Figure 7.3 page 214– Notice Hemoglobin appears to be constructed of 4

Myoglobin strands• Figure 7.4 page 215• Heme is composed of 4 pyrrole rings linked by a-methylene bridges.

• As a whole, the molecule is called Porphin• Each Porphin binds 1 Ferrous Ion (Fe2+)

Structure, cont.

• Oxidation of the iron to Fe3+ destroys biological function

Myoglobin

• Oxygen stored is released to prevent oxygen deprivation

• The oxygen goes to the mitochondria for synthesis of ATP

• Myoglobin is composed of about 75% alpha helixes which is unusually high

• As with most globular proteins, outside is polar and inside is non-polar

His at E7 and F8• The eight helixes are termed A-H starting at the

amino terminal• Notice the Histidine residues at E7 and F8, near the

site of Heme (porphin system) Figure 7.5 page 216• His contains pyrrole like ring as a side chain• Heme binds to myoglobin with the propanate

groups towards the outside (polar) and the nonpolar methyl and vinyl groups towards the inside (see fig 7.4c)

Histidines

• The F8 His actually provides a fifth coordination to the iron providing an actual linkage to the protein. (fig 7.5b)

• The other histidine, E7, lies on the opposite side. (more on this later!)

• Due to the coordination to the F8 His, the iron lies outside the plane of the heme and puckers the heme slightly

Heme binding of O2

• When the iron binds O2, the iron moves closer to the plane, pulling the F8 His with it, thus slightly altering the other residues near the F8 His.

• When the O2 binds, the preferred orientation is with the O-Fe-Heme bond at 90o and the Fe-O-O bond at 121o

Heme binding to CO

• The iron actually binds CO with a similar bond that is 25,000 times stronger!

• However, the C of the CO is sp hybridized and so the Fe-C-O bond should be 180o

• This angle is not allowed due to the presence of the E7 Histidine.

• There is a lone pair of electrons on the Nitrogen that creates steric and electronic repulsions

Heme binding to CO

• As a result, CO is forced to bond like O2 and the C-O-Fe bond is significantly weakened.

• This weakening allows for the great abundance of O2 to predominately bind.

Myoglobin: Storage vs. Transport

• Myoglobin is better for storage than transport• The reasoning is seen in the Oxygen binding

curve. (fig 7.6, page 217)

• Notice how the % saturation doesn’t begin to drop until the PO2 is very low.

• This means that Myoglobin would not release O2 in normal conditions, only in very low levels of O2

Hemoglobin

• The additional properties of hemoglobin that allow it to effectively transport O2 arise from it 4o structure.

• These are referred to as allosteric properties, meaning “other space”

• Hemoglobin is tetrameric and contains 2 pairs of different peptide sub units

• The 1o structure of , ,b g d are highly conserved

Comparisons

• Myoglobin and b-subunits have almost identical 2o and 3o structures

• The a strand is also similar but only contains 7 helices rather than 8

Hemoglobin

• Hemoglobin contains 4 heme groups, therefore it can bind 4 O2 molecules per 1 hemoglobin

• Recall that the binding of O2 slightly changes the structure of the heme and connecting protein

• This slight change allows for the next O2 to bind easier

• This is called cooperative binding

• Cooperative binding helps hemoglobin both load and unload O2

• Cooperative binding is only seen in multimeric proteins.

P50

• P50 is the quantity used to express O2 partial pressure

• P50 is the partial pressure of O2 that half saturates a given hemoglobin

• P50 will vary organism to organism but will always exceed the PO2 in peripheral tissues

Cooperative Binding

• The reason hemoglobin experiences cooperative binding is the large conformational changes that hemoglobin undergoes when O2 is bound

• When O2 in bound, one of the /a b subunits rotates 15o creating a more complex structure

• The relates to profound changes in the 2o, 3o, and 4o structure

Conformations

• Hemoglobin with no O2 bound is said to be in the T (taut) form. (Fig 7.13, page 225)

• Once O2 is bound, the hemoglobin shifts to the R (relaxed) form.

• This conformational shift is what lowers the binding energy for the remaining O2 to bind

• Less conformational change is needed.

The Return Trip

• Hemoglobin not only transports O2 from the lungs to peripheral tissues, but also transports CO2 and H+ from the peripheral tissues back to the lungs

• The CO2 is the by-product of respiration in cells

• The CO2 does not bind to the same sites as O2.

Transport of CO2

• CO2 forms carbamates with terminal amino groups of the proteins of Hemoglobin

• This binding of CO2 changes the charge at the N-terminal from + to –

• This favors additional salt bridges holding hemoglobin together.

Transport of CO2

• Only about 15% of CO2 in transported in this manner

• Most of the rest is transported as bicarbonate• Bicarbonate is formed in erthrocytes by the

hydration of CO2 which is catalyzed by carbonic anhydrase

• Initially, carbonic acid is formed but immediately deprotonates at the pH of the blood

Acidic Environment

• Hemoglobin will bind one H+ for every 2 O2’s released

• This plays a major role in buffering capacity of blood

• The delivery of O2 is enhanced by the acidic environment of the peripheral tissues due to the carbamation stabilizing the T form.

In Lungs

• In the lungs the whole process is reversed!• The reciprocal coupling of H+ and O2 binding is

termed the Bohr effect.

Bohr Effect

• The Bohr Effect is dependent upon the cooperative interactions between the hemes of the tetramer

• Therefore, Myoglobin would not show the Bohr Effect

• So, where do the protons in the Bohr Effect come from and how do they help enhance the release of O2?

You had to ask!!!!

Other Factors

• Release of O2 is also enhanced by the presence of 2,3-biphosphoglycerate (BPG)

• BPG is synthesized in erythrocytes at the low O2 concentrations at peripheral tissues

• BPG helps stabilize the T form of hemoglobin• It binds in the central cavity formed by the

four subunits of hemoglobin (Fig 7.18 p 229)• Only the T form binds BPG

• The space between the H helices of the b chains that line the cavity sufficiently opens only in the T form

• BPG forms salt bridges with the positive charges on the terminal amino groups of both b chains via NA1 (1) and with Lys EF6(82) and His H21 (143).

• These salt bridges must be broken to return to the R state.