Ch. 7 Protein Function and Evolution

Myoglobin and Hemoglobin Both are essential for oxygen need Myoglobin stores O 2 in the muscle Hemoglobin transports O 2 to tissues and CO 2 and H + back to lungs The 2 o structure of hemoglobin resembles myoglobin but the 4 o 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  -methylene bridges. As a whole, the molecule is called Porphin Each Porphin binds 1 Ferrous Ion (Fe 2+ )

Structure, cont. Oxidation of the iron to Fe 3+ 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 O 2 When the iron binds O 2, 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 O 2 binds, the preferred orientation is with the O-Fe-Heme bond at 90 o and the Fe-O-O bond at 121 o

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 180 o 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 O 2 and the C-O-Fe bond is significantly weakened. This weakening allows for the great abundance of O 2 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 P O2 is very low. This means that Myoglobin would not release O 2 in normal conditions, only in very low levels of O 2

Hemoglobin The additional properties of hemoglobin that allow it to effectively transport O 2 arise from it 4 o structure. These are referred to as allosteric properties, meaning “other space” Hemoglobin is tetrameric and contains 2 pairs of different peptide sub units The 1 o structure of  are highly conserved

Comparisons Myoglobin and  -subunits have almost identical 2 o and 3 o structures The  strand is also similar but only contains 7 helices rather than 8

Hemoglobin Hemoglobin contains 4 heme groups, therefore it can bind 4 O 2 molecules per 1 hemoglobin Recall that the binding of O 2 slightly changes the structure of the heme and connecting protein This slight change allows for the next O 2 to bind easier

This is called cooperative binding Cooperative binding helps hemoglobin both load and unload O 2 Cooperative binding is only seen in multimeric proteins.

P 50 P 50 is the quantity used to express O 2 partial pressure P 50 is the partial pressure of O 2 that half saturates a given hemoglobin P 50 will vary organism to organism but will always exceed the P O2 in peripheral tissues

Cooperative Binding The reason hemoglobin experiences cooperative binding is the large conformational changes that hemoglobin undergoes when O 2 is bound When O 2 in bound, one of the  subunits rotates 15 o creating a more complex structure The relates to profound changes in the 2 o, 3 o, and 4 o structure

Conformations Hemoglobin with no O 2 bound is said to be in the T (taut) form. (Fig 7.13, page 225) Once O 2 is bound, the hemoglobin shifts to the R (relaxed) form. This conformational shift is what lowers the binding energy for the remaining O 2 to bind Less conformational change is needed.

The Return Trip Hemoglobin not only transports O 2 from the lungs to peripheral tissues, but also transports CO 2 and H + from the peripheral tissues back to the lungs The CO 2 is the by-product of respiration in cells The CO 2 does not bind to the same sites as O 2.

Transport of CO 2 CO 2 forms carbamates with terminal amino groups of the proteins of Hemoglobin This binding of CO 2 changes the charge at the N-terminal from + to – This favors additional salt bridges holding hemoglobin together.

Transport of CO 2 Only about 15% of CO 2 in transported in this manner Most of the rest is transported as bicarbonate Bicarbonate is formed in erthrocytes by the hydration of CO 2 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 O 2 ’s released This plays a major role in buffering capacity of blood The delivery of O 2 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 O 2 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 O 2 ?

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  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  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.