9.

4: Hemoglobin & myoglobin Prosthetic grp: non-polypeptide moiety

that forms a functional part of a protein.
• Protein – prosthetic grp = apoprotein
Hb: globular, high conc. in RBCs – bind • Protein + prosthetic grp = holoprotein
oxygen in lungs, transport to cells
Heme = protoporphyrin IX with central iron
Transport CO2 & H+ (tissue → lungs), carry
atom: ferrous: Fe2+ (in Hb, myoglobin)
& release NO in blood vessels (potent
vasodilator, inhibits platelet aggregation) Fe2+ can form 5 or 6 covalent bonds
depending on if O2 is bound/ not

Diff forms of human Hb: 4 bonds: to pyrrole N atoms of porphyrin.

Heme prosthetic grp (4): binds oxygen – on Pyrrole rings & connecting C: same aromatic
each polypeptide chain. sys: so same plane, porphyrin bonded Fe2+
also same plane
4 polypeptide chains: α: 141, β:146 a.a.
5th (& 6th) bonds: axis perpendicular to
porphyrin ring.

5th: to N of His imidazole = Proximal

Histidine in globin structures.

In oxy-globins: O2 forms 6th bond (btwn Fe2+

& 2nd His imidazole) = Distal Histidine
Embryo: first few months after conception
In deoxy-globins: 6th position unoccupied
Minor adult Hb: HbA2: 2% normal adult Hb
Heme: in hydrophobic pocket: ~18 aa
residues, ~80 interactions (mostly btwn
apolar side chains & apolar regions of
Myoglobin: single polypeptide & O2-binding porphyrin)
site – captures & releases O2 with changes
in conc. in sarcoplasm of skeletal m. cells Driving force of interaction = expulsion of
water of solvation on association of
Model for what occurs when a single hydrophobic heme with apolar side chains
protomer m. acts alone without in heme pocket
interactions among 4 O2-binding sites in
tetramer m. of Hb. Additional int. in Mb btwn -ve propionate
grps of heme & +ve Arg & His side chains
In Hb: diff in aa seq in this region of heme X-ray crystallography: used to define deoxy
binding site → stabilization of porphyrin & oxy structures of Hb & Mb.
propionates by int. with uncharged histidine
imidazole & water molecules of solvent Sperm whale myoglobin: 1st, horse Hb: 2nd
toward outer surface of molecule. Shows that each globin consists of multiple
α-helical regions connected by turns: allow
folding into characteristic spheroidal shape

Primary, secondary & tertiary structures

All myoglobin chains: 153 a.a: 83 invariant,

15 of these, identical to mammalian Hb.

Variability in seq, but secondary & tertiary

structures – almost identical – same fold
structure of a polypeptide can be arrived at
by diff seq.

All globin chains reversibly bind oxygen

Changes (btwn Mb & Hb): mostly

conservative & preserve physical prop. of
residues.

Changes: partially explained by Mb acting

as a monomer: many of surface residues
interact with water, prevent another myo
m. associating (in Hb: provide H bonds &
non-polar contacts with other subunits in
quaternary structure)

Proximal & distal histidines to heme iron,

hydrophobic heme pockets (form essential
nonpolar contacts with heme): preserved in
all globin chains

Small specific changes in structures →

significant diff in physiological prop. of α, β,
γ, δ Hb chains & Mb chain
~70% residues participate in α-helices of
globin fold → 7 helices in α chain, 8 in β
COMPLETE !!! (p358)
(labelled A – H starting from NH2 terminal)

Interhelical regions: AB, BC, CD…..GH

Nonhelical region at NH2 terminal: NA,

COOH terminal: HC

a.a residues: sequentially numbered from 1

in each lettered helix & interhelical region:
for discussion of particular regions &
residues that hv similar functional/
structural roles in Hb & Mb

Simple equilibrium constant characterizes

O2 binding to Mb.

• [Mb O2]: solution conc. of

oxymyoglobin
• [Mb]: deoxymyoglobin
• [O2]: oxygen
• Keq: equilibrium constant
• Units: moles/ liter

Value of Keq: dependent on pH, ionic

strength, temperature
Binding O2 to Hb – positive cooperativity:
binding of 1st O2 facilitates binding to other
subunits, dissociation of 1st O2 will make
dissociation from other subunits easier.
As O2 is a gas, conc. can be expressed as
pressure in torr (1 torr = I mmHg at 00C & Due to cooperativity, oxygen-saturation
standard gravity) curve for Hb differs from Mb: sigmoidal.

COMPLETE !!! (p359)

P50 = equilibrium constant
Molecular mechanism of cooperativity in Radius of Fe2+ with 6 bonds is reduced – just
O2 binding fits into center of porphyrin without
distortion of conformation.
X-ray diffraction data on deoxyHb: ferrous
atoms - out of the plane of their porphyrins Binding of 1st O2: relatively low affinity
by abt 0.4 – 0.6 Å (0.1nm) constant as steric repulsion btwn porphyrin
& His F8 must be overcome on O2
E. configuration of 5-coordinated ferrous association.
atom in deoxyHb has a slightly larger radius
than distance from center of porphyrin to On binding of O2 to 1st heme, movement of
each pyrrole N: iron can be placed in center Fe2+ into center of porphyrin triggers conf.
of porphyrin only with some distortion of change in other subunits → greater affinity
porphyrin conformation. of O2 to other empty heme sites.

If Fe2+ sits in the plane of the porphyrin, 1. Binding of O2 pulls Fe2+ into porphyrin
proximal His F8 imidazole will interact plane.
unfavorably with porphyrin. 2. Movement of Fe2+ moves bonded
proximal His F8 toward porphyrin.
Unfavorable steric interaction due to 3. Therefore, position of covalently
conformational constraints on His F8 & connected F-helix changes.
porphyrin that forces approach of His F8 4. Interconnected FG corner of subunit
toward porphyrin – less significant changes position: this destabilizes FG
constraints in oxyHb corner’s noncovalent interaction with
With Fe2+ out of plane, conformation is C-helix of adjacent subunit at α1β2 or
unstrained & energetically favored for 5- α2β1 subunit interfaces.
coordinated Fe2+. Conformation becomes 5. Adjacent globin structure changes to a
strained when O2 forms 6th bond. conformation that allows O2 to bind to
its heme with high affinity.
More energetically favorable conformation
for O2-bonded Fe2+: Fe2+ is within plane So, FG to C inter-subunit contacts act as a
switch btwn 2 diff arrangements btwn FG
Binding energy of O2 overcomes repulsive corner of one subunit & C-helix of adjacent.
interaction btwn His F8 & porphyrin, Fe2+
moves into plane: most thermodynamically New conformation allows His F8 residues in
stable position for oxy-heme: 6-bonded Fe2+ deoxyHb chains to approach porphyrins on
having one axial ligand on either side of O2 association with less steric repulsion.
plane of porphyrin: steric repulsion of one Ionic interactions that stabilize deoxy-
axial ligand balanced by 2nd on opp. side. conformation are broken by conf. change
induced: oxy-conf. in other subunits can
form more easily.
DeoxyHb: tense/ T conformational state According to law of mass action, increasing
[H+] on right side forces eq. to left:
Oxy form with higher O2 affinity (X150-300, increases [deoxy-Hb] & [free O2]
depending on the solution conditions):
relaxed/ R conformational state. Increased cellular metabolic activity
increases release of carbonic acid from CO2
Allosteric mechanism describes how initial & lactic acid: so, increases [H+].
binding of O2 to a heme pushes molecular
conf. from T to R state. Increase in acidity forces oxy → deoxy-Hb,
O2 delivered to metabolizing cells.

Feedback loop: cellular metabolic acid

Hb facilitates transport of CO2 & NO waste signals increase in O2 dissociation for
NO = vasodilator, Hb delivers it to blood continuation of cellular metabolism.
vessel walls in tissues Protons dissociate (T → R) when acid side
T to R conformational equilibrium of Hb chains of Hb are more acidic in R than T:
controls delivery of O2, CO2 & NO to pKa of these grps should be lower in R.
appropriate sites. His-146(β): major contributor to H+
Due to changes in pKa of side chain aa grps dissociation on T → R
btwn T & R conf. equilibrium is regulated by In T: +ve charge on His-146(β) side chain
[H+]: changes in pH link delivery of O2 & NO imidazolium is in an ion pair with -ve charge
to tissues, transport of CO2 away. of carboxylate side chain of Asp-94 (β):
stabilizes +ve charge

Decrease in pKa of acid groups with change Stabilized imidazolium tends to retains +ve
from T to R conformation releases protons charge, has a higher than normal pKa (~7.7)

Release of protons (T → R) at blood pH = T → R breaks this ion pair, Asp & His side
alkaline Bohr effect: physiological chains at new positions in R – no longer
importance strongly interact: loss of opp. charge
interaction reverts His-146(β) imidazolium
to more normal pKa (~7.3)

Change in His-146(β) pKa to a more acidic

n = equivalents of protons released when value causes dissociation of protons at 7.4
one molecule of deoxy-Hb → oxy-Hb: 1.2- (pH of blood)
2.7, depending on solution conditions, [Cl-],
[2,3-bisphosphoglycerate]
~50% protons that dissociate come from Carbonic acid spontaneously dissociates
His-146(β): others from acid grps that into HCO3- & H+.
similarly change pKa with T → R
HCO3- diffuses out of RBC, carried to lung by
At more acidic/ lower pH (higher [H+]), Hb plasma: isohydric transport (CO2 from
dissociates its O2 more easily, curve shifted tissues to lungs in plasma as HCO3-): ~70-
to right. Higher P50 value at more acidic pH: 80% produced CO2
represents poorer O2 affinity as oxygen-
binding sites are 50% saturated at higher H+ bind to Hb, force Hb(O2)4 to dissociate
p(O2) conc. O2: diffuses out of RBC to cells.

Therefore, Hb acts as buffer of blood pH by

binding H+ produced by metabolizing cells.

Protons bind side chains: eg: imidazole of

His-146(β), deoxy H+Hb transported to lungs
in RBC.

High pO2 conc converts H+Hb (T) to Hb(O2)4

(R), protons dissociate, combine with HCO3-:
diffuse back to RBC to form carbonic acid,
carbonic anhydrase converts it to CO2 + H2O

CO2 diffuses to lung alveoli, expired into air

CO2 & O2 transport: linked by Bohr-effect

protons.

CO2 produced by metabolizing cells diffuses

into surrounding blood → RBC. Converted
by hydration reaction to carbonic acid
(enzyme: carbonic anhydrase)
2nd mechanism of CO2 transport: 15 – 20 % Formed in a minor pathway for glucose
thru carbamino-Hb: non-enzymatic reaction metabolism, present in small amounts in all
of CO2 with NH2 terminal amino grps of Hb cells
chains: also produces H+
In RBC: this pathway = highly active, [BPG] ~
+
Excess H produced binds to Hb, stabilizes equimolar to [Hb].
deoxy form, promoting release & delivery of
O2 to actively metabolizing cells. BPG binds to only deoxy (T) form, stabilizes
T, increases conc. relative to R.
In lung, high pO2 conc generates Hb(O2)4
with dissociation of H+ - increase in H+ BPG dissociates as T → R
forces dissociation of carbamino grp,
releases CO2 – expired from lung.
Increased [BPG] force equilibrium to left,
shift saturation plot to right with increase in
P50.

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

diphosphoglycerate (DPG) in RBC: imp
modulator of Hb equilibrium

Single BPG molecule binds within pocket

formed at β1-β2-subunit interface in T conf.

This site contains 8 +ve grps: (His-143(β),

Lys-82(β), His-2(β), NH2-terminal
ammonium residues from both β chains)
BPG charge -5, strongly attracted to these Molar conc of NO in blood = 1/70th [Hb]:
+ve charges in binding site. low conc is physiologically significant due to
potent biological activity of X-S-NO
R: size of binding pocket decreased, BPG
unable to fit easily into binding pocket: so, NO – first captured by Fe2+ at heme - T, then
T → R causes BPG dissociation. transferred to sulfhydryl of β-chain cystein-
93 residues - R.
Conditions that cause hypoxia (deficient
oxygen): anemia, smoking, high altitude: Heme iron preferentially binds NO when in
increase BPG levels in RBC. T conformation, transferred to βCys93 when
Hb is in R conf.
Hyperoxia conditions lower BPG levels
When R → T to deliver O2 to tissues, NO
Changes in RBC BPG levels: slow, occur over transferred from βCys93 to small S-XH
hours & days to compensate for chronic molecules (eg: glutathione): net effect:
changes in pO2 levels conversion by Hb of unstable free plasma
NO to stable X-S-NO.

Hb delivers NO to capillary wall of tissues: In T: found outside, accessible to

promotes O2 delivery glutathione m.: allows transnitrosylation to
occur.
Hb reversibly binds NO (potent vasodilator,
short lifetime in blood) – sequesters it from In R: βCys93 -SH: pointed toward heme iron,
rapid destruction. inaccessible to outside solvent.

Hb releases NO by transferring it to small Distance in R btwn heme iron & βCys93:

sulfhydryl (X-SH) molecules as Hb changes optimal for transfer of NO from heme to
from R → T. βCys93 -SH.

Common X-SH molecule = glutathione. Thus, conf changes in Hb, induced by

changes in pO2 btwn lungs & tissues,
X-S-NO compound continues to stabilize NO regulate NO uptake & release from βCys93.
against degradation & allows efficient
delivery of bioactive NO equivalent (X-S- Net effect: delivery of bioactive NO to tissue
NO) to NO receptors in cells of blood vessel capillaries under low oxygen tension
wall: promotes relaxation of vascular wall.

Relaxation facilitates transfer of gasses

btwn blood & cells of tissues.