BCHE5180 Hemoglobin handout (Exam2)

negative heterotropic

2,3-BPG is a _____ ______ modulator. It binds to the central cavity of hemoglobin but only in the T state (Fig. 8). In the R-state the cavity is too small.

homotropic allosteric modulator

A _______ is a ligand for its target protein, as well as a regulatory molecule of the proteins affinity. Both types of modulator may be either an activator or an inhibitor of the protein or enzyme.

heterotropic allosteric modulator

A _________ is a regulatory molecule that is not the ligand. Both types of modulator may be either an activator or an inhibitor of the protein or enzyme.

2,3‑bisphosphoglycerate (2,3-BPG); 2,3-BPG

A really important modulator is _______, synthesized by erythrocytes. ______ is a negative heterotropic modulator. It binds to the central cavity of hemoglobin but only in the T state (Fig. 8). In the R-state the cavity is too small.

1.0 Å Other helices could move more, the same amount or less

Approximately how far in Angstroms does Helix F appear to shift when oxygen binds to heme? What will happen to other helices in the subunit when helix F moves?

α

Before the conformation changes, only the heme groups within the ___ subunits are accessible for binding O2. (before R state is reached)

carbaminohemoglobin; T; salt bridges; lungs

Bohr Effect: CO2 itself will also bind the hemoglobin. It binds as carbamate to the amino group at the N-terminus, forming ____. This further stabilizes the ___ state of hemoglobin through the formation of _________. Hemoglobin will bind large amounts of CO2 in the tissues, up to 40% present, and releases them in the ____ when the pH changes back to 7.6. It can be said that hemoglobin transports both O2 to the tissues and CO2 to the lungs.

T; lowering

Bohr Effect: H+ protons protonate residues stabilizing the __ state of hemoglobin, ____ binding affinity of O2.

lower; low

Carbon dioxide is produced by metabolizing tissues and is hydrated to from bicarbonate by the blood bicarbonate buffer system. Protons (H+) are a byproduct of the CO2 conversion to bicarbonate by carbonic anhydrase. As a result the pH in the tissues is _____: 7.2 vs. 7.6 in the lungs. Protons bind to several amino acid residues in hemoglobin when H+ concentrations are higher (___ pH environments). A major contributor to the Bohr effect is when H+ bind to His146 on the β subunits, protonating the residues.

Heme is more flat and the iron ion is in the plane of the tetrapyrrole ring.

Describe the overall shape of the heme group (porphyrin-Fe2+-ring) with oxygen bound to Fe2+.

Heme is bent (puckered) and the iron ion sticks out on one site.

Describe the overall shape of the heme group, (porphyrin-Fe2+-ring) without oxygen bound to Fe2+.

θ is a fraction. When all sites are occupied: θ= 4/4 = 1 When θ = 0.5, Only 2 O2 are present. The hemoglobin would still in be in the R state.

Explain why θ ranges from 0 to 1. What does your group think θ = 0.5 means at the molecular level for a sample of hemoglobin molecules? Draw pictures to convey your ideas

binds; releases

Hemoglobin _____ O2 strongly when the pO2 is high (lungs) and ____ it when the pO2 is low (tissues).

heme c; His

Hemoglobin and myoglobin bind oxygen through a heme group, _____, sequestered into a hydrophobic pocket within the protein structure. _____ is a prosthetic group, an essential part of the protein structure. It is covalently bound to 2 _____ residues

tetramer; four

Hemoglobin is an _______ consisting of 4 polypeptide chains (Fig. 5). Each polypeptide chain in hemoglobin contains a single heme group and are similar to each other and myoglobin. For all purposes one can say that hemoglobin is made up of 4 myoglobins. Each subunit is therefore able to bind one O2 molecule. SO hemoglobin can bind ___ total Oxygen. The four subunits have extensive interactions creating a cavity in the center of the hemoglobin molecule.

relaxed; tense

Hemoglobin is an allosteric protein. Allosteric proteins have unique ligand binding properties, where the binding of one ligand to a binding site effects the binding properties of another site on the protein. In all cases the protein subunits exist in one of two conformations, tensed (T) or relaxed (R), and that ___ subunits bind substrate more readily than those in the ____ state.

The levels should be higher. The answer might seem counter intuitive but remember that hemoglobin already binds all oxygen available in the blood when is passes through the lungs. Higher levels of 2,3-BPG, which will increase the relative amounts of O2 released in the tissues.

How do the levels of O2 in the atmosphere compare at sea level versus high altitude? Predict the blood levels of BPG for people who live at high altitudes, like Denver. How will those levels of BPG help their hemoglobin function better?

0.6 Å

How far in Angstroms does the F8 histidine nitrogen appear to shift when oxygens binds to the Fe2+?

1 subunit, α

How many components does myoglobin have, and what are they?

4 subunits, two α and two β

How many subunits does hemoglobin have? What are their conventional names?

hydroxyl radical (OH●); Fenton's Fe2+ + H2O2 → Fe3+ + OHˉ + OH●

Hydrogen peroxide is also able to oxidize Fe2+ and produce a _______. This reaction is called _____ reaction:

Oxygen binds to heme groups. Myoglobin has only 1 heme. Hemoglobin has four (one per subunit).

Identify the oxygen binding sites on myoglobin and hemoglobin. How many oxygens can one subunit of hemoglobin bind? How many oxygens can one molecule of hemoglobin bind?

O2; electron; N-ligands

In deoxyhemoglobin (T state) the heme is not flat but puckered and the Fe2+ is positioned just outside of the plane of the porphyrin ring. When _____ binds to Fe2+, the ring structure gets a more planar conformation and the iron ion is now positioned in the plane of the ring. Note that the Fe2+ is protected from oxidation to the 3+ state because of the _____ donating character of the _________ coordinating the iron-ion and the stabilization of the O2 molecule.

T; β; α; R

In the __ state the ___ subunits are inaccessible to O2 meaning that the first O2 molecule has to bind to an ___ subunit. Within the subunits, the binding and the change in heme structure drags the His F8 residue with it as well as the whole F helix and the 2 connecting segments EF and FG (Fig. 7). These movements are transmitted to the subunit interfaces triggering conformational readjustments between the four subunits to maintain most of the interactions. As a result, there is now an increased O2 binding affinity for all subunits: the ___ state. Before the conformation changes, only the heme groups within the α subunits are accessible for binding O2.

R

In the __ state, all four sites are accessible for O2 binding and the four binding sites on the hemoglobin molecule will quickly saturate with oxygen

T

In the lungs the high concentration of oxygen will push the equilibrium towards the ___-state and all binding sites will be fully occupied

T; low

In the tissues some oxygen will be released due to the lower oxygen concentration but the binding of H+, CO2 and BPG will pull hemoglobin towards the ___-state with ___ affinity for oxygen causing the release of pretty much all oxygen bound dependent on how much the tissues need at that moment.

1

Myoglobin can bind ____ oxygen

8 helical ; bends; hydrophobic; E and F; F ; His F8

Myoglobin consists of a single polypeptide chain that contains ___ _____ regions that are connected by small ____ (Fig. 4). The helical regions are named A through H and the connecting bends are named for the 2 helices it is between (ex. AB, BC, EF). The heme group is bound to a _____ pocket created between the ___ and ___ helices. The His residue bound to Fe2+ is in the ___helix and is also known as ___

Heme C

O2 binding site for myoglobin and hemoglobin

The P50 value is larger, which represents weaker binding affinity of O2

On Model 1, draw a new binding curve that is shifted to the right. Label the P50 on the new curve. How does the new value of P50 for your new curve compare to the original P50 value? What does the shifted/"new" hemoglobin curve indicate in regard to hemoglobin and oxygen binding affinity?

ATP; proteins that transport oxygen!

Organisms utilize oxygen to drive _____ synthesis. In multicellular organisms, this process is conducted in every cell within every tissue creating a need for oxygen to be distributed throughout the whole organism continuously. (cellular respiration). To meet this need a circulatory system was employed to facilitate oxygen transport. Capillaries widely dispersed in the body facilitate gas exchange between blood and interstitial tissue. There was one problem with the circulatory system though, oxygen (O2) dissolved poorly in blood and hardly migrates into tissue so getting it into the blood to be transported was a major hurdle. Nature came up with several solutions to this problem. which was.....

free iron and heme-Fe2+ Fe2+ + O2 → Fe3+ + O2●ˉ

Oxidation of ____ and ____ produces an oxygen radical, Superoxide (O2●ˉ):

positive homotropic

Oxygen is an example of a ______ _______ effector to hemoglobin. When O2 binds to a heme group within hemoglobin it causes a conformational change from T to R state, which results in increased binding affinity for future O2 molecules.

hydrogen peroxide (H2O2): 2 O2●ˉ + 2 H2O → O2 + H2O2 + 2 OHˉ

Oxygen radicals undergo dismutation extremely rapidly forming ________: (first step formation of HO2)

oxidative stress

Reactive Oxygen Species are involved in some cell signaling processes but the accumulation of them results in _______ which can lead to DNA damage and mutation, lipid peroxidation, inactivation of iron-sulfur cluster containing enzymes, and disulfide bond formation. These symptoms cause physiological dysfunction and eventually result in cell death.

methemoglobin; methemoglobin; Superoxide (O2●ˉ):

Sequestration of heme minimizes Fe2+ oxidation to ferric cation (Fe3+). When oxidized, _____ can't bind O2 rendering the erythrocyte functionally useless. Erythrocytes produce _______ reductase to convert Fe3+ back to Fe2+ restoring its O2 binding ability. The protection of heme-Fe2+ from oxidation is very important. Oxidation of free iron and heme-Fe2+ produces an oxygen radical, _____.

T

Structure of 2,3-Bisphosphoglycerate (2,3-BPG) and it binding site in hemoglobin. 2,3-BPG will only bind when the protein is in the ____ state when the cavity between the subunits is big enough

Reactive Oxygen Species (ROS)

Superoxide, hydrogen peroxide and the hydroxyl radical belong to the group of _______. which are dangerous to the body. The body is equipped with defense mechanisms such as superoxide dismutase, catalase, and antioxidants to control the levels of radicals.

lowers; right

The binding of BPG stabilizes the T state of hemoglobin through electrostatic interactions. These interactions maintain the rotational interactions between α and β subunits characterizing the T state of hemoglobin. BPG ____ the binding affinity for O2 but only in the tissues when the T state is induced. It shifts the binding curve to the _______ ensuring O2 release in tissues to maintain O2 saturation in tissues. In the absence of 2,3-BPG the binding curve of hemoglobin strongly resembles that of myoglobin (green curve in Figure 9, labeled 'Stripped Hb').

R; T

The equilibrium will shift to the ___ state in the lungs and the ___ state in the tissues.

T(ense) ; R(elaxed); quaternary; T ; deoxyhemoglobin; R; oxyhemoglobin

The fact that hemoglobin is a multimeric protein forms the basis for the changed oxygen binding properties. Hemoglobin exists in 2 different states, the _____ state and the ______ state. These states have different ____ structures and relate to the oxygenation of hemoglobin. When there is no O2 present hemoglobin exists in the ___state, as ________. Once O2 binds hemoglobin transitions to the ___ state, becoming ________.

higher

The fetal hemoglobin form (Hbf) differs from the adult form in that the His143 residue in the β chains is replaced with a Ser residue. By binding less 2,3-BPG Hbf has a ____ affinity for oxygen than Hba which ensures oxygen transfer through the placenta and adequate oxygen saturation in the tissues of the growing fetus.

1) that when parasites invade Hbs containing erythrocytes and start using up the oxygen the cells will deform and the body targets the cells for destruction, and 2) that erythrocytes containing Hbs produce more O2●ˉ which possibly stunts or kills the parasite.

The genetic trait for Hbs was maintained despite the Sickle-cell Anemia disease because individuals with Hbs molecule are able to survive the disease Malaria. The precise mechanism of resistance is unknown but two prevailing theories are:

lung; saturated; tissues; release; saturated; zero

The important consequence of this is that when hemoglobin travels through the capilaries in the ___ it will become completely _____ with oxygen, while when it travels through the capilaries in the _____ it can ____ pretty much all bound oxygen (red curve in Figure 6). Myoglobin is different in that it is still highly _____ even at lower oxygen concentration and will only release the O2 when the O2 concentration is close to _____ (blue curve in Figure 6).

When the pO2 is low (in the tissues), some O2 is released and some of the hemoglobin returns to the T state. That opens up a binding site for 2,3-BPG which binds and pulls the equilibrium even more to the left (picture) releasing even more O2 and so on. This process is only possible in the tissues. In the lungs all enzyme is in the R-state and 2,3-BPG cannot bind.

The molecule 2,3-BPG binds to the deoxy form of hemoglobin in the central cavity. Explain why that makes sense for oxygen delivery and why BPG is said to stabilize the deoxy state.

θ; cooperative binding

The positive change in hemoglobin saturation, ___, as more O2 is present is called _________.

His; hydrogen bond

The sequestration of heme within the protein is important for its ability to bind O2. When heme is sequestered into myoglobin or hemoglobin a ___ residue is positioned close by so that it can provided a _____ to the non-iron-bound oxygen atom within O2 increasing the favorability of O2 in myoglobin/hemoglobin

sigmoidal; lower; accelerated; allosteric

The structural differences between myoglobin and hemoglobin result in different binding affinities for O2. In hemoglobin, however, the O2 binding curve is _____ (Fig. 6). In low concentrations of O2, hemoglobin binding of O2 is ____ than that of myoglobin. The O2 binding curve of hemoglobin reveals a region of ____ binding affinity for O2 when the first O2 molecules bind. This is the ______ nature of hemoglobin.

hyperbolic; a single O2 molecule is bound.

The structural differences between myoglobin and hemoglobin result in different binding affinities for O2. The O2 binding curve for myoglobin is ____, O2 is bound up at low concentrations of O2 in the environment, as it is in muscle tissues (Fig. 6). The single heme group in myoglobin also means that total saturation is reached after ....

allosteric ; allosteric

This change in configuration and the change in binding affinity for molecular oxygen is called the _____ effect. Hemoglobin is an ____ protein.

Bohr

Two more modulators are important for the transport of oxygen by hemoglobin: H+ and CO2. The effect of these on hemoglobin is better known as the ____ Effect.

hemoglobin; Myoglobin; Myoglobin

Vertebrates use erythrocytes (red blood cells) for oxygen transport. They do so by producing a protein called ____ to bind O2 and carry it through the circulatory system into capillaries for partial gas exchange with carbon dioxide (CO2). Oxygen is picked up in the lungs and transported to actively metabolizing tissues - such as muscles - where it is released. This transport system in the circulatory system works well between the tissues and the lungs but tissues require an additional protein to facilitate O2 transport deeper into the tissue. _____ fills this role. ______ is a smaller protein that is primarily found in muscle that functions as an oxygen transporter.

To pick up molecular oxygen in the lungs, transport it in the bloodstream and release it in the tissues.

What is the function of hemoglobin? Discuss with your group how the shape of this curve communicates that hemoglobin effectively delivers oxygen from the lungs to the tissues.

0.6 Å

When oxygen binds to the heme group, by how many Angstroms does the Fe2+ appear to shift?

P50

When θ = 0.5, the value of pO2 is defined as

4

When θ equals 1.0 how many oxygen molecules are bound to one hemoglobin molecule?

Modulators or effectors

____ or ____ will affect the equilibrium between both R and T states for hemoglobin allosteric proteins

pO2

_____ refers to the actual amount of O2 present in the solution.

Positive allosteric modulation

_______ (also known as allosteric activation) occurs when the binding of one ligand enhances the attraction between substrate molecules and other binding sites (equilibrium shifts toward the R state).

Hemerythrin; Hemerythrin; Hemerythrin; hydroperoxide; iron; hydroperoxide *contains Fe (iron)

_______ is an oligomeric protein responsible for oxygen transport in the marine invertebrate phyla of Sipunculids and Priapulids (unsegmented marine worms), and Brachiopods (looks like a clam but is not). ________ is essentially colorless when deoxygenated, but turns a violet-pink in the oxygenated state. Note that 'heme' is derived from the Greek word for blood. The mechanism of dioxygen binding is unusual. Most O2 carriers operate via formation of dioxygen complexes, but ________ holds the O2 as a _____. The site that binds O2 consists of a pair of ____ centers (Fig. 2). The iron atoms are bound to the protein through the carboxylate side chains of a glutamate and aspartate as well as through five histidine residues. The uptake of O2 by hemerythrin is accompanied by two-electron oxidation of the diferrous center to produce a _____ (OOH−) complex.

Negative allosteric modulation

________ (also known as allosteric inhibition) occurs when the binding of one ligand decreases the affinity for substrate at other active sites (equilibrium shifts towards the T state).

Cooperativity; Oxygen affinity; Allosterism

________ - interaction of ligands and binding affinity ________ - strength of attraction for O2 in hemoglobin ______ - A change in the activity and conformation of an enzyme resulting from the binding of a compound at a site on the enzyme other than the active binding site Hemoglobin is an allosteric protein that exhibits cooperativity between subunits for O2 binding. BPG is a negative effector of hemoglobin and reduces the oxygen affinity when BPG is bound.

Hemocyanins; copper; color; Cu(I); Cu(II)

_________ are proteins that transport oxygen throughout the bodies of Mollusca (includes: snails, octopuses, squid, clams, scallops, oysters, and chitons) and Arthropoda (arachnids, crustaceans and insects). These metalloproteins contain two ____ atoms that reversibly bind a single oxygen molecule (Fig. 1). The proteins are suspended directly in the hemolymph which is analogous to blood in vertebrates. Oxygenation causes a ____ change between the colorless _____ deoxygenated form and the blue _____ oxygenated form.

Although the changes in pO2 are similar the changes in θ are much larger. At lower values of pO2 the increase in oxygen binding is smaller, while at higher pO2 values the increase in oxygen binding is much larger. This is an example of cooperative binding. This effect is mainly detected in the 'lower part' of the curve (θ between 0 and 0.5). At even higher pO2 values the enzyme becomes saturated and the increase levels off again.

describe how the value of θ changes with increasing pO2 levels. (Use the term oxygen binding in your answer.)

homotropic; homotropic

he binding of oxygen at one site affects the binding properties of oxygen at another site on the protein. This type of cooperativity is ___ since oxygen binding at one site is affecting oxygen binding at another site. The term ____ is used because the same ligand, oxygen, is involved at the multiple sites.

high; low

hemoglobin has a greater affinity for oxygen when the partial pressure of oxygen is ____ and has a lower affinity when the partial pressure of oxygen is ____

pO2; θ

oxygen binding curve for hemoglobin: X-axis: Y-axis:

lower

pH in lungs is ____ due to release of CO2 and removal of HCO3ˉ. Hb becomes deprotonated and more O2 can bind. The opposite happens in the tissues when CO2 levels are very high.