Voet D., Voet Ju.G. Biochemistry

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, increases the amount of O

that hemoglobin unloads

in the capillaries (Fig. 10-10). Similar increases in BPG

concentration occur in individuals suffering from disor-

ders that limit the oxygenation of the blood (hypoxia),

such as various anemias and cardiopulmonary insuffi-

ciency.

b. Fetal Hemoglobin Has a Low BPG Affinity

The effects of BPG also help supply the fetus with

oxygen. A fetus obtains its O

from the maternal circulation

via the placenta.This process is facilitated because fetal he-

moglobin (HbF) has a higher O

affinity than does mater-

nal hemoglobin (HbA; recall that HbF has the subunit

composition ␣

␥

, in which the ␥ subunit is a variant of

HbA’s ␤ subunit; Section 7-3C). BPG occurs in about the

same concentrations in adult and fetal erythrocytes but

binds more tightly to deoxyHbA than to deoxyHbF; this ac-

counts for HbF’s greater O

affinity. In the next section we

shall develop the structural rationale for the effect of BPG

and for the other aspects of O

binding.

2 STRUCTURE AND MECHANISM

The determination of the first protein X-ray structures,

those of sperm whale myoglobin by John Kendrew in 1959

and of human deoxyhemoglobin and horse methemoglo-

bin by Max Perutz shortly thereafter, ushered in a revolu-

tion in biochemical thinking that has reshaped our under-

standing of the chemistry of life. Before the advent of

protein crystallography, macromolecular structures, if they

were considered at all, were thought of as having a rather

hazy existence of uncertain biological significance. How-

ever, as the elucidation of macromolecular structures has

continued at an ever quickening pace, it has become clear

that life is based on the interactions of complex, structurally

well-defined macromolecules.

The story of hemoglobin’s structural determination is a

tale of enormous optimism and tenacity. Perutz began this

study in 1937 at Cambridge University as a graduate student

of J. D. Bernal (who, with Dorothy Crowfoot Hodgkin, had

taken the first X-ray diffraction photographs of hydrated

protein crystals in 1934). In 1937, the X-ray crystal structure

determination of even the smallest molecule required many

months of hand computation, and the largest structure yet

determined was that of the dye phthalocyanin, which has 40

nonhydrogen atoms. Since hemoglobin has ⬃4500 nonhy-

drogen atoms, it must have seemed to Perutz’s colleagues

that he was pursuing an impossible goal. Nevertheless, the

laboratory director, Lawrence Bragg (who in 1912, with his

father William Bragg, had determined the first X-ray struc-

ture, that of NaCl), realized the tremendous biological sig-

nificance of determining a protein structure and supported

the project.

It was not until 1953 that Perutz finally hit on the method

that would permit him to solve the X-ray structure of hemo-

globin, that of isomorphous replacement. Kendrew, a col-

league of Perutz, used this technique to solve the X-ray

structure of sperm whale myoglobin, first at low resolution

in 1957, and then at high resolution in 1959. Hemoglobin’s

greater complexity delayed its low-resolution structural

determination until 1959, and it was not until 1968, over 30

years after he had begun the project, that Perutz and his as-

sociates obtained the high-resolution X-ray structure of

horse methemoglobin. Those of human and horse deoxy-

hemoglobins followed shortly thereafter.Since then, the X-

ray structures of hemoglobins from numerous different

species, from mutational variants, and with different bound

ligands have been elucidated. This, together with many of-

ten ingenious physicochemical investigations, has made he-

moglobin the most intensively studied, and perhaps the

best understood, of proteins.

In this section, we examine the molecular structures of

myoglobin and hemoglobin and consider the structural ba-

sis of hemoglobin’s oxygen-binding cooperativity, the Bohr

effect, and BPG binding.

A. Structure of Myoglobin

Myoglobin consists of eight helices (labeled A–H) that are

linked by short polypeptide segments to form an ellipsoidal

Section 10-2. Structure and Mechanism 331

1.0

0.8

0.6

0.4

0.2

0 20 40 60 80 100

Venous

Arterial

4500 m

Arterial

sea level

0.38

0.3

0.37

= 31 torr (high BPG)

= 26 torr (normal BPG)

(torr)

Figure 10-10 The O

-dissociation curves of blood adapted to

sea level (black curve) and to high altitude (red curve). Between

the sea level arterial and venous pO

values of 100 and 30 torr,

respectively, Hb normally unloads 38% of the O

it can maximally

carry. However, when the arterial pO

drops to 55 torr, as it does

at an altitude of 4500 m, this difference is reduced to 30% in

nonadapted blood. High-altitude adaptation increases the BPG

concentration in erythrocytes, which shifts the O

-dissociation

curve of Hb to the right.The amount of O

that Hb delivers to

the tissues is thereby restored to 37% of its maximum load.

JWCL281_c10_323-358.qxd 2/24/10 1:57 PM Page 331

molecule of approximate dimensions 44 ⫻ 44 ⫻ 25 Å (Fig.

10-11; see also Fig. 8-39).The helices range in length from 7

to 26 residues and incorporate 121 of myoglobin’s 153

residues (Table 10-1). They are largely ␣ helical but with

some distortions from this geometry such as a tightening of

the final turns of helices A, C, E, and G to form segments of

helix.

In a helix numbering convention peculiar to globins,

residues are designated according to their position in a he-

lix or interhelical segment. For example, residue B5 is the

fifth residue from the N-terminus of the B helix and

residue FG3 is the third residue from the N-terminus in

the nonhelical segment connecting helices F and G. The

nonhelical N- and C-terminal segments are designated NA

and HC, respectively. The usual convention of sequentially

numbering all amino acid residues from the N-terminal

residue of the polypeptide is also used, and often both con-

ventions are used together. For example, Glu EF7(83) of

human Mb is the 83rd residue from its N-terminus and the

7th residue in the nonhelical segment connecting its E and

F helices.

The heme is tightly wedged in a hydrophobic pocket

formed mainly by helices E and F but which includes

contacts with helices B, C, G, and H as well as the CD and

FG segments. The fifth ligand of the heme Fe(II) is His

F8, the proximal (near) histidine. In oxyMb, the Fe(II) is

positioned 0.22 Å out of the heme plane on the side of

the proximal His and is coordinated by O

with the bent

geometry shown in Fig. 10-12. His E7, the distal (distant)

histidine, hydrogen bonds to the O

. In deoxyMb, the

sixth liganding position of the Fe(II) is unoccupied be-

cause the distal His is too far away from the Fe(II) to co-

ordinate with it. Furthermore, the Fe(II) has moved to a

point 0.55 Å out of the heme plane. Other structural

changes in Mb on changing oxygenation states consist of

small motions of various chain segments and slight read-

justments of side chain conformations. By and large, how-

ever, the structures of oxy- and deoxyMb are nearly super-

imposable.

B. Structure of Hemoglobin

The hemoglobin tetramer is a spheroidal molecule of di-

mensions 64 ⫻ 55 ⫻ 50 Å. Its two ␣␤ protomers are sym-

metrically related by a twofold rotation (Fig. 10-13; see also

Fig. 8-64). The tertiary structures of the ␣ and ␤ subunits are

remarkably similar, both to each other and to that of Mb

(Figs. 10-11 and 10-13), even though only 18% of the corre-

sponding residues are identical among these three

polypeptides (Table 10-1) and there is no D helix in hemo-

globin’s ␣ subunit. Indeed, the a and b subunits in the

tetramer are related by pseudo- (inexact) 2-fold rotations so

that the subunits occupy the vertices of a tetrahedron

(pseudo-D

symmetry; Section 8-5B).

The polypeptide chains of Hb are arranged such that

there are extensive interactions between unlike subunits. The

␣

–␤

interface (and its ␣

–␤

symmetry equivalent) in-

volves 35 residues, whereas the ␣

–␤

(and ␣

–␤

) interface

involves 19 residues. These associations are predominantly

332 Chapter 10. Hemoglobin: Protein Function in Microcosm

Table 10-1 The Amino Acid Sequences of the ␣ and ␤ Chains of Human Hemoglobin and of Human Myoglobin

a,b

Helix Boundaries A1 A16 B1 B16 C7 D1 D7

C1 E1

1 5 10 15 20 25 30 35 40 45 50 55 60 65

Hb ␣..................V– LSPADKTNVKAA WGKVGAHAGEY GAEA LERMFLSFPTTKTYFPHF– DL SH–––––G SAQVKGHGKK VADALT

1 5 10 15 20 25 30 35 40 45 50 55 60 65 70

Hb ␤..................VHLTPEEKSAVTALWGKV––NVDE V GGE A LGR L LVVY PWT QR FFESF GD L STPDAVMG NPK VKA HGKK V LGA FS

1 5 10 15 20 25 30 35 40 45 50 55 60 65 70

Mb ....................G–LSDGEWQLVLNVWGKVEADI PGHGQEVLIRL FK GHPET LEKFDKFKHLKSEDEMKA SEDLKKHGAT VLTA LG

Figure 10-11 Structure of sperm whale myoglobin. Its 153 C

␣

positions are numbered from the N-terminus and its eight helices

are sequentially labeled A through H.The last half of the EF

corner is now regarded as a turn of helix and is therefore

designated the F¿ helix.The heme group is shown in red. Also see

Fig. 8-39. [Illustration, Irving Geis. Image from the Irving Geis

Collection, Howard Hughes Medical Institute. Reprinted with

permission.] Based on an X-ray structure by John Kendrew,

MRC Laboratory of Molecular Biology, Cambridge, U.K. PDBid

1MBN.]

See Kinemage Exercise 6-1

152

151

153

137

139

138

140

143

142

141

144

146

145

148

150

149

133

135

10 9

114

115

120

119

121

122

124

126

130

108

107

105

101

100

123

⬘

JWCL281_c10_323-358.qxd 8/10/10 9:40 AM Page 332

hydrophobic in character, although numerous hydrogen

bonds and several ion pairs are also involved (Section

10-2C). In contrast, contacts between like subunits,

␣

–␣

and ␤

–␤

, are few and largely polar in character.

This is because like subunits face each other across an

⬃20-Å-diameter solvent-filled channel that parallels

the 50-Å length of the exact 2-fold axis (Figs. 8-64

and 10-13).

a. Oxy- and Deoxyhemoglobins Have Different

Quaternary Structures

Oxygenation causes such extensive quaternary struc-

tural changes to Hb that oxy- and deoxyHb have different

crystalline forms; indeed, crystals of deoxyHb shatter on

exposure to O

. The crystal structures of hemoglobin’s oxy

and deoxy forms therefore had to be determined inde-

pendently. The quaternary structural change preserves he-

moglobin’s exact 2-fold symmetry and takes place entirely

across its a

–b

(and a

–b

) interface. The ␣

–␤

(and ␣

–␤

)

contact is unchanged, presumably as a result of its more ex-

tensive close associations. This contact provides a conven-

ient frame of reference from which the oxy and deoxy con-

formations may be compared. Viewed in this way,

oxygenation rotates the ␣

␤

dimer ⬃15° with respect to

the ␣

␤

dimer (Fig. 10-14), so that some atoms at the ␣

–␤

interface shift by as much as 6 Å relative to each other

(compare Figs. 10-13a and 10-13b).

The quaternary conformation of deoxyHb is named the

T state (T for “tense”). That of oxyHb, which is essentially

independent of the ligand used to induce it (e.g., O

, met,

CO, CN

⫺

, and NO hemoglobins all have the same quater-

nary structure), is called the R state (R for “relaxed”). Sim-

ilarly, the tertiary conformational states for the deoxy and

liganded subunits are designated as the t and r states,

respectively.The structural differences between the quater-

nary and tertiary conformations are described in the fol-

lowing subsection in terms of hemoglobin’s O

-binding

mechanism.

Section 10-2. Structure and Mechanism 333

The residues have been aligned in structurally analogous positions.The blue boxes shade the residues that are identical in both Hb chains, the purple

boxes shade the residues that are identical in both Hb chains and in Mb, and the dark purple boxes shade residues that are invariant in all vertebrate Hb

and Mb chains (Thr C4, Phe CD1, Leu F4, His F8, and Tyr HC2).

The first and last residues in helices A–H are indicated, whereas the residues between helices constitute the intervening “segments.”The refined Hb

structure reveals that much of what is designated the EF segment is really helical in both chains: It encompasses residues EF4–F2 and is designated the

F¿ helix.

Source: Dickerson, R.E. and Geis, I., Hemoglobin, pp. 68–69, Benjamin/Cummings (1983).

NAV AHVDDM PNALSA LSD L HA

D GLAHL DNLKGTFA TLSELHC

G I LKKKGHHEA E I K P L AQSHA

Hb ␣...

Hb ␤...

Mb.......

HKLRVDPV NFKLLSHCLLVT L

D KLHV DPE NFRLLGNVLVCVL

TKH K IPVKYLEFISECIIQVL

AAHL PAE F T PAV HASLDK FL A S

AHHFGKEF T PPVQAAYQKVV AG

QSKHPGDFG ADA QGAMNK ALEL

VSTVLTSKY R

V ANA L AHKYH

F RKDM A S N YKELGFQG

E19 F1 F9G1 G19 H1 H19

H21

H26

70 75 80 85 90 95 100 105 110 115 120 125 130 135 140

75 80 85 90 95 100 105 110 115 120 125 130 135 140 145

75 80 85 90 95 100 105 110 115 120 125 130 135 140 145 150

Figure 10-12 The heme complex in oxyMb. In the upper

drawing, atoms are represented as spheres of van der Waals radii.

The lower drawing shows the corresponding skeletal model with

a dashed line representing the hydrogen bond between the distal

His and the bound O

. [After Phillips, S.E.V., J. Mol. Biol. 142,

544 (1980). PDBid 1MBO.]

See Kinemage Exercise 6-1

His E7

Phe CD1

Val E11

His F8

JWCL281_c10_323-358.qxd 2/24/10 1:57 PM Page 333

C. Mechanism of Oxygen-Binding Cooperativity

The positive cooperativity of O

binding to Hb arises from

the effect of the ligand-binding state of one heme on the

ligand-binding affinity of another. Yet the distances of 25 to

37 Å between the hemes in an Hb molecule are too large

for these heme–heme interactions to be electronic in char-

acter. Rather, they are mechanically transmitted by the pro-

tein. The elucidation of how this occurs has motivated much

of the structural research on Hb for the past four decades.

X-ray crystal structure analysis has provided “snapshots”

of the R and T states of Hb in various states of ligation but

does not indicate how the protein changes states. It is difficult

to determine the sequence of events that result in such trans-

formations because to do so requires an understanding of

the inner workings of proteins that is presently lacking. It is

as if you were asked to explain the mechanism of a compli-

cated mechanical clock from its out-of-focus photographs

when you had only a hazy notion of how gears, levers, and

springs might function. Nevertheless, largely on the basis of

the X-ray structures of Hb, Perutz formulated the following

mechanism of Hb oxygenation, the Perutz mechanism.

334 Chapter 10. Hemoglobin: Protein Function in Microcosm

Figure 10-13 The X-ray structures of (a) deoxyHb and

(b) oxyHb as viewed down their exact 2-fold axes. The C

␣

atoms,

numbered from each N-terminus, and the heme groups are

shown.The Hb tetramer contains a solvent-filled central channel

paralleling its 2-fold axis, whose flanking ␤ chains draw closer

together on oxygenation (compare the lengths of the double-

headed arrows). In the deoxy state, His FG4(97)␤ (small single-

headed arrow) fits between Thr C6(41)␣ and Pro CD2(44)␣

(lower right and upper left).The relative movements of the two

␣␤ protomers on oxygenation (large gray arrows in Part b) shift

His FG4(97)␤ to a new position between Thr C3(38)␣ and Thr

C6(41)␣. See Fig. 8-64 for a similarly viewed space-filling model

of deoxyHb. [Illustration, Irving Geis. Image from the Irving

Geis Collection, Howard Hughes Medical Institute. Reprinted

with permission.] Based on X-ray structures by Max Perutz,

MRC Laboratory of Molecular Biology, Cambridge, U.K.

PDBids (a) 2DHB and (b) 2MHB.]

See Kinemage Exercises

6-2 and 6-3

(a)

JWCL281_c10_323-358.qxd 8/10/10 9:40 AM Page 334

Section 10-2. Structure and Mechanism 335

DeoxyHb

C3(38)

␣

C6(41)␣

FG4(97)␤

CD2(44)␣

C3(38)␣

C6(41)␣

FG4(97)␤

CD2(44)␣

␣

␤

(a)

OxyHb

(b)

␤

15⬚

C3(38)␣

FG4(97)␤

C6(41)␣

CD2(44)␣

C3(38)␣

FG4(97)␤

C6(41)␣

CD2(44)␣

OxyHb

(b)

15⬚

␣

␤

15⬚

Figure 10-14 The major structural differences between the

quaternary conformations of (a) deoxyHb and (b) oxyHb. On

oxygenation, the ␣

␤

(shaded) and ␣

␤

(outline) dimers move,

as indicated on the right, as rigid units such that there is an ⬃15°

off-center rotation of one protomer relative to the other that

preserves the molecule’s exact 2-fold symmetry. Note how the

Figure 10-13 (continued)

position of His FG4␤ (pentagons) changes with respect to Thr

C3␣,Thr C6␣, and Pro CD2␣ (yellow dots) at the ␣

–␤

and

␣

–␤

interfaces.The view is from the right side relative to that in

Fig. 10-13. [Illustration, Irving Geis. Image from the Irving

Geis Collection, Howard Hughes Medical Institute. Reprinted

with permission.]

(b)

JWCL281_c10_323-358.qxd 8/10/10 9:40 AM Page 335

a. The Movement of Fe(II) Into the Heme Plane

Triggers the T S R Conformational Shift

In the t state, the Fe(II) is situated ⬃0.6 Å out of the

heme plane on the side of the proximal His because of a

pyramidal doming of the porphyrin skeleton and because

the Fe¬N

porphyrin

bonds are too long to allow the Fe to lie

in the porphyrin plane (Figs. 10-15 and 10-16). The change

in the heme’s electronic state on binding O

, however,

causes the doming to subside and the Fe¬N

porphyrin

bonds

to contract by ⬃0.1 Å. Consequently, on changing from the

t to the r state, the Fe(II) moves to the center of the heme

plane (Fig. 10-16) where O

can coordinate it without steric

interference from the porphyrin.The Fe’s movement drags

the proximal His along with it, which tilts the attached

F helix and translates it ⬃1 Å across the heme plane

(Fig. 10-16). This lateral translation occurs because, in the

t state, the imidazole ring of the proximal His is oriented

such that its direct movement of ⬃0.6 Å toward the heme

plane would cause it to collide with the heme (Figs. 10-15

and 10-16); however, the F helix shift reorients the imida-

zole ring, thereby permitting the Fe(II) to move into the

heme plane. In addition, in the t state of the ␤ but not the ␣

subunits, Val E11 partially occludes the O

-binding pocket

so that it must be moved aside before O

binding can occur.

b. The ␣

–␤

and ␣

–␤

Contacts Have Two

Stable Positions

As we saw above, the difference between hemoglobin’s

R and T conformations occurs mainly in the ␣

–␤

(and the

336 Chapter 10. Hemoglobin: Protein Function in Microcosm

Figure 10-16 Triggering mechanism for the T S R transition

in Hb. In the T form (blue), the Fe is ⬃0.6 Å above the mean

plane of the domed porphyrin ring. On assuming the R form

(red), the Fe moves into the plane of the now undomed

porphyrin, where it can readily bind O

, and, in doing so, pulls

the proximal His F8 and its attached F helix with it.The Fe¬O

bond is thereby strengthened because of the relaxation of the

steric interference between the O

and the heme. See

Kinemage Exercise 6-4 and the Animated Figures

Leu F4 (83)

His

F8 (87)

Leu H19

(136)

Leu F7

Leu FG3

(91)

Val FG5 (93)

Tyr C7 (42)

(86)

Phe CD4

(46)

His E7

(58)

Leu G8

(101)

Val E11

(62)

Lys E10

(61)

contact

Porphyrin Porphyrin

Heme

Helix F

Leu F7

Leu F4 Leu FG3

Val FG5

His F8

0.6 A

Figure 10-15 The heme group and its

environment in the unliganded ␣ chain

of human Hb. Only selected side chains

are shown and the heme D propionate

group is omitted for clarity. The F helix

runs along the left side of the

drawing.The close contact

between the proximal His

and the heme group that

inhibits oxygenation

of t-state hemes is

indicated by a

dashed red line.

[After Gelin, B.R.,

Lee, A.W.N., and Karplus, M.,

J. Mol. Biol. 171, 542 (1983).

PDBid 2HHB.]

See

Kinemage Exercise 6-4

JWCL281_c10_323-358.qxd 2/24/10 1:58 PM Page 336

symmetrically related ␣

–␤

) interface, which consists of

the C helix and FG segment of ␣

, respectively, contacting

the FG segment and C helix of ␤

. The quaternary change

results in a 6-Å relative shift at the ␣

C–␤

FG interface

(Fig. 10-14). In the T state, His FG4(97)␤ is in contact with

Thr C6(41)␣ (Figs. 10-13a and 10-17a), whereas in the R

state it is in contact with Thr C3(38)␣, one turn back along

the C helix (Figs. 10-13b and 10-17b). In both conforma-

tions, the “knobs” on one subunit mesh nicely with the

“grooves”on the other (Fig.10-17).An intermediate position,

Section 10-2. Structure and Mechanism 337

Figure 10-17 The ␣

C–␤

FG interface of Hb in (a) the T state

and (b) the R state. The upper drawings show the C helix in

ribbon form (purple) and its contacting portion of the FG region

in ball-and-stick form colored according to atom type (C green,

N blue, and O red).The dots outline the contacting van der

Waals surfaces and are also colored according to atom type.The

lower drawings are the corresponding schematic diagrams of the

␣

C–␤

FG contact. On a T S R transformation, this contact

snaps from one position to the other with no stable intermediate

(a) T State (deoxy)

oxygenation

(b) R State (oxy)

Asp G1 (99)

Tyr C7

(42)

Asn G4 (102)

Asp G1 (94)

His FG4

–

Thr C3 38

Thr C6 41

Pro CD2 44

Thr C3 38

Thr C6 41

Pro CD2 44

His FG4

(note how, in both conformations, the knobs formed by the side

chains of His 97␤ and Asp 99␤ fit between the grooves on the C

helix formed by the side chains of Thr 38␣, Thr 41␣, and Pro

44␣).The subunits are joined by different hydrogen bonds in the

two quarternary states. Figures 10-13 and 10-14 provide

additional structural views of these interactions. [Based on X-ray

structures by Giulio Fermi, Max Perutz, and Boaz Shaanan,

MRC Laboratory of Molecular Biology, Cambridge, U.K.

PDBids (a) 2HHB and (b) 1HHO.]

See Kinemage Exercise 6-5

JWCL281_c10_323-358.qxd 6/3/10 10:27 AM Page 337

however, would be severely strained because it would

bring His FG4(97)␤ and Thr C6(41)␣ too close together

(knobs on knobs). Hence these contacts, which are joined by

different but equivalent sets of hydrogen bonds in the two

states (Fig. 10-17), act as a binary switch that permits only

two stable positions of the subunits relative to each other. In

contrast, the quaternary change causes only a 1-Å shift at

the ␣

FG–␤

C contact, so its side chains maintain the same

associations throughout the change. These side chains

therefore act as flexible joints or hinges about which the a

and b

subunits pivot during the quaternary change.

c. The T State Is Stabilized by a Network of Salt

Bridges That Must Break to Form the R State

The R state is stabilized by ligand binding. But in the

absence of ligand, why is the T state more stable than the

R state? In the electron density maps of R-state Hb, the C-

terminal residues of each subunit (Arg 141␣ and His 146␤)

appear as a blur, which suggests that these residues are free

to wave about in solution.Maps of the T form,however,show

these residues firmly anchored in place via several intersub-

unit and intrasubunit salt bridges, which evidently help stabi-

lize the T state (Fig. 10-18). The structural changes accompa-

nying the T S R transition tear away these salt bridges in a

process driven by the Fe¬O

bonds’ energy of formation.

d. Hemoglobin’s O

-Binding Cooperativity Derives

from the T S R Conformational Shift

The hemoglobin molecule resembles a finely tooled

mechanism that has very little slop. The binding of O

re-

quires a series of tightly coordinated movements:

1. The Fe(II) of any subunit cannot move into its

heme plane without the reorientation of its proximal His

so as to prevent this residue from bumping into the por-

phyrin ring.

2. The proximal His is so tightly packed by its surround-

ing groups that it cannot reorient unless this movement is

accompanied by the previously described translation of the

F helix across the heme plane.

3. The F helix translation is only possible in concert

with the quaternary shift that steps the ␣

C–␤

FG contact

one turn along the ␣

C helix.

4. The inflexibility of the ␣

–␤

and ␣

–␤

interfaces re-

quires that this shift simultaneously occur at both the ␣

–␤

and the ␣

–␤

interfaces.

Consequently, no one subunit or dimer can greatly

change its conformation independently of the others. In-

deed, the two stable positions of the a

C–b

FG contact limit

the Hb molecule to only two quaternary forms, R and T.

We are now in a position to structurally rationalize hemo-

globin’s O

-binding cooperativity. Any deoxyHb subunit

binding O

is constrained to remain in the t state by the T

conformation of the tetramer. However, the t state has re-

duced O

affinity, most probably because its Fe¬O

bond is

stretched beyond its normal length by the steric repulsions be-

tween the heme and the O

and in the b subunits, by the need

to move Val E11 out of the O

-binding site. As more O

bound to the Hb tetramer,this strain,which derives from the

Fe¬O

bond energy, accumulates in the liganded subunits

until it is of sufficient strength to snap the molecule into the

R conformation. All the subunits are thereby converted to the

r state whether or not they are liganded. Unliganded subunits

in the r state have an increased O

affinity because they are al-

ready in the O

-binding conformation. This accounts for the

high O

affinity of nearly saturated Hb.

338 Chapter 10. Hemoglobin: Protein Function in Microcosm

Figure 10-18 Networks of salt bridges and hydrogen bonds in

deoxyHb. These bonds, which involve the last two residues of (a)

the ␣ chains and (b) the ␤ chains, are all ruptured in the T S R

transition.The two groups that participate in the Bohr effect by

Val

Lys

Val

Asp

Tyr

Arg

N-terminal

127

126

141

140

C-terminal

(a) α Chains

Val

Lys

Asp

Tyr

His

146

145

C-terminal

(b) β Chains

becoming partially deprotonated in the R state are indicated by

white plus signs. [Illustration, Irving Geis. Image from the Irving

Geis Collection, Howard Hughes Medical Institute. Reprinted

with permission.]

JWCL281_c10_323-358.qxd 8/10/10 9:41 AM Page 338

e. Hemoglobin’s Sigmoidal O

-Binding Curve Is a

Composite of Its Hyperbolic R- and T-State Curves

The relative stabilities of the T and R states, as indicated

by their free energies, vary with fractional saturation

(Fig. 10-19a). In the absence of ligand, the T state is more

stable than the R state, and vice versa when all ligand-

binding sites are occupied.The formation of Fe¬O

bonds

causes the free energy of both the T and the R states to de-

crease (become more stable) with oxygenation, although

the rate of this decrease is smaller for the T state as a result

of the strain that liganding imposes on t-state subunits. The

R 12 T transformation is, of course, an equilibrium process,

so that Hb molecules, at intermediate levels of fractional

saturation (1, 2, or 3 bound O

molecules), continually in-

terconvert between the R and the T states.

The O

-binding curve of Hb can be understood as a com-

posite of those of its R and T states (Fig. 10-19b). For pure

states, such as R or T, these curves are hyperbolic because lig-

and binding at one protomer is unaffected by the state of

other protomers in the absence of a quaternary structural

change.At low pO

’s,Hb follows the low-affinity T-state curve

and at high pO

’s, it follows the high-affinity R-state curve.At

intermediate pO

’s, Hb exhibits an O

affinity that changes

from T-like to R-like as pO

increases. The switchover results

in the sigmoidal shape of hemoglobin’s O

-binding curve.

D. Testing the Perutz Mechanism

The Perutz mechanism is a description of the dynamic be-

havior of Hb that is largely based on the static structures

of its R and T end states. Accordingly, without the direct

demonstration that Hb actually follows the postulated

pathway in changing conformational states, the Perutz

mechanism must be taken as being at least partially con-

jectural. Unfortunately, the physical methods that can fol-

low dynamic changes in proteins are, as yet, incapable of

providing detailed descriptions of these changes. Never-

theless, certain aspects of the Perutz mechanism are sup-

ported by static measurements, as is described below and

in Section 10-3.

a. C-Terminal Salt Bridges Are Required

to Maintain the T State

The proposed function of the C-terminal salt bridges in

stabilizing the T state has been corroborated by chemically

modifying human Hb. Removal of the C-terminal Arg 141␣

(by treating isolated ␣ chains with carboxypeptidase B fol-

lowed by reconstitution) drastically reduces the cooperativ-

ity of O

binding (Hill constant of 1.7, reduced from its nor-

mal value of 2.8). Cooperativity is abolished by the further

removal of the other C-terminal residue, His 146␤ (Hill con-

stant of ⬃1.0). Apparently, in the absence of its C-terminal

salt bridges, the T form of Hb is unstable. Indeed, human

deoxy-Hb, with its C-terminal residues removed, crystal-

lizes in a form very similar to that of normal human oxyHb.

b. Fe¬O

Bond Tension Has Been

Spectroscopically Demonstrated

If movement of the Fe into the heme plane on oxygena-

tion is mechanically coupled via the proximal His to the

T S R transformation, then conversely, forcing oxyHb into

the T form must exert a tension on the Fe, through the prox-

imal His, that tends to pull the Fe out of the heme plane. Pe-

rutz demonstrated the existence of this tension as follows.

Section 10-2. Structure and Mechanism 339

Figure 10-19 Free energy and saturation curves for O

binding

to hemoglobin. (a) The variation of the free energies of

hemoglobin’s T and R states with their fractional saturation,

In the absence of O

the T state is more stable, and when

saturated with O

the R state is more stable. The free energy of

both states is reduced with increasing oxygenation as a

consequence of O

liganding.The Fe(II)¬O

bonding is more

(a) (b)

R State

T State

R State

T State

Free

energy,

0.5

1.0

exergonic in the R state than it is in the T state, however, so that

the relative stabilities of these two states reverse order at

intermediate levels of oxygenation. (b) The sigmoid O

-binding

curve of Hb (purple) is a composite of its hyperbolic R-state

(red) and T-state (blue) binding curves: It is more T-like at lower

values and more R-like at higher pO

values.

JWCL281_c10_323-358.qxd 2/24/10 1:58 PM Page 339

IHP’s six phosphate groups cause it to bind to deoxyHb with

much greater affinity than does BPG (the structural basis of

BPG binding to Hb is discussed in Section 10-2F); the pres-

ence of IHP therefore tends to force Hb into the T state.

Conversely, nitric oxide (NO) binds to Hb far more strongly

than does O

and thereby tends to force Hb into the R state.

Spectroscopic analysis indicates the consequences of simul-

taneously binding both NO and IHP to Hb:

1. The NO, as expected, pulls the Fe into the plane of

the heme.

2. The IHP forces the Hb molecule into the T state,

which through the “gears and levers” coupling the 4° and 3°

conformational changes, pulls the proximal His in the

opposite direction, away from the Fe.

The bond between the proximal His and the Fe lacks the

strength to withstand these two opposing “irresistible”

forces; it simply breaks. The spectroscopic observation of

this phenomenon therefore confirms the existence of the

heme–protein tension predicted by the Perutz mechanism.

c. Detaching the Proximal His from the F Helix

Eliminates Most Cooperativity

In a further experimental investigation of the origin of

cooperativity in hemoglobin, Chien Ho mutagenically

changed the proximal His residue to Gly on only the ␣ sub-

units, on only the ␤ subunits, and on both the ␣ and ␤ sub-

units. The missing imidazole ring of the proximal His was

then replaced by imidazole (which a variety of evidence in-

dicates ligands the heme Fe as does the proximal His). This,

in effect, detaches the proximal His from the protein,

thereby cutting the covalent bond that, according to the Pe-

rutz model, links the ligand-induced movement of the Fe

into the heme plane to the accompanying movement of he-

lix F. In all three cases, this proximal detachment, in agree-

ment with the Perutz model, significantly increases hemo-

globin’s ligand-binding affinity,reduces its cooperativity, and

prevents its T S R quaternary switch. However, these mu-

tant hemoglobins exhibit a small amount of residual cooper-

ativity, suggesting that the heme groups also communicate

via pathways that do not require covalent coupling between

the F helix and the proximal His. These pathways may in-

volve movements of protein groups in contact with the heme

(see Figs. 10-12 and 10-15) in response to the subsidence of

heme doming on ligand binding. They may also involve

movements of the distal His residues of the ␣ and ␤ subunits,

and/or the movement of Val E11 of the ␤ subunits, all of

whose side chains must move aside when ligand binds to Hb.

E. Origin of the Bohr Effect

The Bohr effect, hemoglobin’s release of H

⫹

on binding O

is also observed when Hb binds other ligands. It arises from

pK changes of several groups caused by changes in their

local environments that accompany hemoglobin’s T S R

transition. The groups involved include the N-terminal

amino groups of the ␣ subunits and the C-terminal His of

the ␤ subunits. These have been identified through chemi-

cal and structural studies, and their quantitative contribu-

tions to the Bohr effect have been estimated.

Reaction of the ␣ subunits of Hb with cyanate results in

the specific carbamoylation of the N-terminal amino

groups (Fig. 10-20). When such carbamoylated ␣ subunits

are mixed with normal ␤ subunits, the resulting reconsti-

tuted Hb lacks 20 to 30% of the normal Bohr effect. The

reason for this is seen on comparing the X-ray structure

of deoxyHb with that of carbamoylated deoxyHb. In

deoxyHb, a Cl

⫺

ion binds between the N-terminal amino

group of Val 1␣

and the guanidino group of Arg 141␣

(the

C-terminal residue; Fig. 10-18a). This Cl

⫺

is absent in car-

bamoylated deoxyHb. It is also absent in normal R-state

Hb because its C-terminal residues are not held in place by

salt bridges (which partially accounts for the preferential

binding of Cl

⫺

to deoxyHb; Section 10-1C). N-Terminal

amino groups of polypeptides normally have pK’s near 8.0.

On deoxyHb ␣ subunits, however, the N-terminal amino

group is electrostatically influenced by its closely associ-

ated Cl

⫺

to increase its positive charge by binding protons

more tightly, that is, to increase its pK. Since at the pH of

blood (7.4) N-terminal amino groups are normally only

partially charged, this pK shift causes them to bind signifi-

cantly more protons in the T state than in the R state.

The Hb ␤ chain also contributes to the Bohr effect. Re-

moval of its C-terminal residue, His 146␤, reduces the Bohr

effect by 40%. In normal deoxyHb, the imidazole ring of

His 146␤ associates with the carboxylate of Asp 94␤ on the

same subunit (Fig. 10-18b) to form a salt bridge that is ab-

sent in the R state. Proton NMR measurements indicate

that formation of this salt bridge increases the pK of the

imidazole group from 7.1 to 8.0. This effect more than ac-

counts for His 146␤’s share of the Bohr effect.

We have not yet accounted for about 30 to 40% of the

Bohr effect. It is largely due to small contributions from the

numerous surface-exposed His residues whose environ-

ments are altered on hemoglobin’s T S R transition [since

His is the only residue with an intrinsic pK (6.04) in the

physiological range, small changes in its pK will signifi-

cantly alter the number of protons it binds]. Indeed, NMR

measurements by Ho indicate that the T S R transition in-

duces small shifts in the pK’s of these various His residues,

although, interestingly, some of these shifts are in the direc-

tion that diminishes the magnitude of the Bohr effect.

340 Chapter 10. Hemoglobin: Protein Function in Microcosm

Figure 10-20 Reaction of cyanate with the unprotonated

(nucleophilic) forms of primary amino groups. At physiological

pH’s, N-terminal amino groups, which have pK’s near 8.0, readily

react with cyanate. Lys ε-amino groups (pK ⬇ 10.8), however, are

fully protonated under these conditions and are therefore

unreactive.

R NH

+ N

–

R NH

Terminal

amino

group

Cyanate Carbamoylated

terminal amino

group

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