Urry D.W. (Ed.) What Sustains Life? : Consilient Mechanisms for Protein-Based Machines and Materials

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8.3 The Electron Transport Chain: Protein Machines as Redox-driven Proton Pumps

375

brane. Introduction of the hydrophobic con-

silient mechanism to proton gating, whereby

charge disrupts hydrophobic association and

opens aqueous channels, occurs as the studies on

elastic-contractile model protein reviewed in

Chapter 5 become viewed in the light of the

molecular structure of Complex III. This is

discussed in section

8.3.4.4

with consideration of

the Hunte et al.^^ structure of Complex III that

includes diffraction-detectable water molecules.

83.4.3 Consilient Mechanisms for

Electron Transfer/Proton Transport Reside

in the Movement of the Rieske Iron

Protein FeS Center

8.3.4.3.1

Molecular Structures That Show

Relocation of the Globular Domain of the

Rieske Iron Protein

Molecular structures of Complex III are often

obtained with the FeS center of the RIP in posi-

tion for docking at the Qo site of the second

monomer. This orientation, obtained using stig-

matellin and antimycin inhibitors, is shown

for the monomer in stereo view in Figure 8.14A

with charged residues in white, neutral residues

in light gray, aromatic residues in black, and

other hydrophobic residues in gray. Thus, the

darker regions indicate the more hydrophobic

domains of the protein subunit. Zhang et al.^^

determined a crystal structure with the FeS

center of the RIP at the position of the

cytochrome Ci site of the second monomer, as

similarly shown in stereo view in Figure 8.14B.

Thus,

electron transfer is estabhshed in this

unique case to be one of protein domain relo-

cation subsequent to addition of the electron to

the redox center. How does this dynamic

mechanical event occur? As discussed immedi-

ately below, this utilizes both hydrophobic

and elastic consihent mechanisms by interlink-

ing hydrophobic association/dissociation and

elastic extension/relaxation.

8.3.4.3.2

Consideration of Hydrophobic

Association on Docking of the Rieske FeS

Center at Qo and Cytochrome Ci Sites

The two docking sites for the FeS center of the

RIP are shown in space-filling representation in

Figure 8.15. By the use of white for charged

residues and gray to black for increasingly

hydrophobic residues, it becomes apparent by

inspection of the involved surface areas,

without calculation of the Gibbs free energy

for hydrophobic association, AGHA, that the Qo

site is much more hydrophobic than the

cytochrome Ci site. The hydrophobicity of the

globular component of the RIP that associates

with either the Qo site or the cytochrome Ci

site is even more apparent. The very dark tip,

shown in end view and in side view in Figures

8.16A,B, respectively, makes clear that the

interaction of the FeS center with the Qo site

would clearly be dominated by hydrophobic

association.

8.3.4.3.3

Stretched Tether Connecting the

Membrane Anchor to the Globular

Component of the Rieske Iron Protein When

Hydrophobically Associated at the Qo Site

The membrane anchor of the RIP associates at

an angle with other transmembrane helices that

define it as part of one monomer. It then crosses

over by means of a tether from one monomer

to interact with the other. The hydrophobic

globular component of the RIP that contains

the FeS center has its most favorable hydropho-

bic association at the Qo site of the adjacent

monomer. As shown in Figure 8.17 and also

notable in Figure 8.14, to achieve this most

favorable

AGHA

a section of the tether becomes

stretched to near full extension.

The two issues that now require examination

are whether the visually extended tether is

kinetically free and whether the chain does in

fact contract (shorten) as the FeS center moves

from the Qo site of cytochrome b to the heme

of cytochrome Ci.

8.3.4.3.4 Is the Tether of the Rieske Iron

Protein a Free Standing (Kinetically Free)

Chain When the FeS Center Is at the

Qo Site?

Useful elastic force develops at the fixed ends

of a chain segment when the motion within the

backbone of the chain segment decreases as the

result of a deformation such as an increase in

376

8. Consilient Mechanisms for Protein-based Machines of Biology

Extended tether

to FeSXJenter

Heme c

stigmatelli

at Qo site

Heme b

^ Rieske

FeS Center

-Matrix end of Rieske Iron

^rotein membrane anchor

Relaxed tether

to FeS Center

Heme c

Heme b

Rieske

FeS Center

FIGURE 8.14. Stereo view of the monomer of

Complex

III:

the cytochrome bci complex of chicken.

Shown are Hgands plus the Rieske Iron Protein,

where the inhibitors stigmatellin and antimycin are

used to show

FeS

center relocation on reduction. The

Rieske Iron Protein anchors in one monomer and

then reaches across to accept an electron at its FeS

center from the Qo site of the second monomer. (A)

With inhibitors, the FeS center is in position, with the

tip of the globular component of the Rieske Iron

Protein pointing directly downward at the level of

the Qo site, where it is to be reduced by the second

component of the functional homodimer. (Prepared

using the crystallographic results of Zhang et al.^^ as

obtained from the Protein Data Bank, Structure File

3BCC.) (B) Without inhibitors, the FeS center is in

position with the tip of the globular component of

the Rieske Iron Protein directed out of the plane at

the level of the heme

to reduce cytochrome

the second unseen component of the functional

homodimer. (Prepared using the crystallographic

results of Zhang et

al.^^

as obtained from the Protein

Data Bank, Structure File IBCC.) Note that the

tether connecting the membrane anchor to the glob-

ular component of the Rieske Iron Protein subunit

is extended in part B when the FeS center is at the

Qo site and relaxed in part A when the FeS site is at

the level of the heme

site.

8.3 The Electron Transport Chain: Protein Machines as Redox-driven Proton Pumps

377

eme d

tigmatellin

site

•Heme Cj

site

-F169 fulcrum

ocation of

Q^^

site

Cyt. b^

FIGURE

8.15.

Stereo view of yeast cytochromes b and

Ci of Complex III showing Qo and heme Ci binding

sites for the FeS center of the Rieske Iron Protein.

Neutral residues are given in light gray, aromatic

residues in black, other hydrophobic residues in gray,

and charged residues in white in order to show visu-

ally the relative hydrophobicity of the two

sites.

Note

at the

site that ubiquinol would reside at the base

hydrophobic pit into which the hydrophobic FeS

tip of the Rieske Iron Protein fits (see Figure 8.16),

whereas the heme

site is not as hydrophobic. Also

note that the hydrophobic residues L263 and V264

constitute a raised rim of the pit over which the FeS

center must pass in order to dock at the heme

site.

(A) Sites in the presence of inhibitors where the FeS

center resides at the Qo site with the configuration

shown in Figure 8.16. (Prepared using the crystallo-

graphic results of Zhang et

al.^^

as obtained from the

Protein Data Bank, Structure File 3BCC.) (B) Sites

in the absence of inhibitors where the FeS center

resides at the heme

site (Prepared using the crys-

tallographic results of Zhang et

al.^^

as obtained from

the Protein Data Bank, Structure File IBCC.)

378

8. Consilient Mechanisms for Protein-based Machines of Biology

e-S Center

f^3%

j^**^

•

^ti^ ^Bf

FIGURE 8.16. Stereo view of the Rieske Iron Protein

(RIP) component of the monomer of Complex III:

cytochrome bci complex of

chicken.

Shown in space-

filling representation is the RIP with neutral residues

in light gray, hydrophobic residues in gray, aromatic

residues in black, and charged residues in white.

(Prepared using the crystallographic results of

Zhang et al.^^ as obtained from the Protein Data

Rieske

Fe-S Center

Bank, Structure File 3BCC.) (A) Stereo view show-

ing the very hydrophobic tip at the FeS center of the

globular component of the RIP. (B) Stereo side view

of the globular component of the RIP showing the

hydrophobicity becoming greatest at the tip that

would form a particularly favorable hydrophobic

association in the pit of the Qo site.

the distance between the fixed ends. This

requires that the chain segment be sufficiently

free from intermolecular interaction so that the

backbone motions are responsive to the limita-

tions posed by deformation. A kinetically free

and sterically unrestrained chain is the best way

to achieve this structural requirement. Thus an

examination of the structure in space-filling

representation with all chains present, that

is,

the complete functional homodimer, provides

the most direct opportunity to determine

whether a chain segment may be applying a

force at its ends.

By ribbon representation, Figure 8.18B pro-

vides a stereo view perpendicular to the twofold

axis of an expanded section of dimer in which

the tethers are seen crossing from one monomer

to the other. These tethers that connect the RIP

membrane anchor to the globular component

for both monomers are shown totally free

standing, without any interchain interactions.

The cross-eye stereo view in space-filling repre-

8.3 The Electron Transport Chain: Protein Machines as Redox-driven Proton Pumps

379

sentation in Figure 8.18A of the same expanded

section shows the tether in the foreground to be

free standing without contact with other protein

chain segments. Thus, the tether is kinetically

free,

being constrained in its motion only by the

extending forces at its ends. This is exactly as

required to provide an entropic elastic force by

the mechanism of the damping of internal chain

dynamics on extension.

8.3.4.3.5

Is the Tether Contracted When the

FeS Center of the Rieske Iron Protein

Resides at the Heme Ci Site?

For the tether to be responsible for the motion

of the FeS center of the globular component of

the Rieske center from the Qo site to the heme

Ci site it must have contracted; the length of

the tether must have shortened. This question

Kinetically free

and stretched

paired tethers to

Iron Proteins

(res.

A86 to V94)

'F169

as fulcrum

eS center

membrane anchor

(a-helical)

for Iron Protein

Kinetically free

and stretched

paired tethers to

Iron Proteins

(res.

A86 to V94)

F169

as fulcrum

eS center

membrane anchor

(a-helical)

for Iron Protein

FIGURE 8.17. Stereo view of the components of the

homodimer of yeast cytochrome bci:—one molecule

of cytochrome b in space-filling representation, one

molecule of cytochrome

in space-filling represen-

tation, and both of the FeS-containing Reiske Iron

Proteins present are shown with one in gray back-

bone representation and the other in space-filling

representation. As usual for the space-filling repre-

sentations, neutral residues are in light gray,

hydrophobic residues in gray, aromatic residues in

black and charged residues in

white.

The FeS centers

of the Rieske Iron Proteins are at their Qo sites on

the cytoplasmic sides of the molecules of cytochrome

(A) View perpendicular to twofold axis to show

both tethers, one in backbone representation on the

left and the other in space-filling representation on

the right. (B) View rotated to be seen from above

with heme c included. (Prepared using the crystallo-

graphic results of Lange and Hunte^^ as obtained

from the Protein Data Bank, Structure File, IKYO.)

380

8. Consilient Mechanisms for Protein-based Machines of Biology

can be addressed using the crystal structure of

cytochrome bci from chicken that is available

for both states of Qo site and heme Ci site occu-

pancy.^^ In the most appropriate backbone rep-

resentation in Figure 8.19B, the tether is shown

in its extended state by noting the distance from

residues A66 to K77, whereas the A66 to K77

end-to-end distance is shortened in Figure

8.19A.

This shortening is also apparent in

Figure 8.14A,B- As shown in Figure 8.19, the

tether is most dramatically shortened for the

segment from A66 to 174. Thus, the second

criterion for the tether to function as a con-

tractile element to achieve domain movement

has been met.

As with the elastic-contractile model pro-

teins discussed in Chapter 5, favorable

hydrophobic association (in this case of RIP

globular protein tip with the Qo site) stretches

interconnecting chain segments. Thus, the

answer to the question asked by Crofts et al.^°

in the title of their article, becomes clear: "Inter-

actions of quinone with iron-sulfur protein of

the bci complex: Is the mechanism spring-

1 4\

Free standing

tether (in gray)

to Iron Protein

Lipid layer of

mitochondrial

inner membrane

Kinetically free

and stretched

paired tethers to

Iron Proteins

(res.

A86 to

V94)

Lipid layer of

mitochondrial

inner membrane

membrane anchor

(a-helical)

for Iron Protein

FIGURE 8.18. An expanded stereo view of the com-

plete yeast Complex

III:

dimer of the cytochrome bci

complex. Identified are the tethers of Rieske Iron

Protein connecting the membrane anchor to the

globular component. The purpose is to demonstrate

that the tether sequence from A66 to V94 is free

standing in the structure and would, therefore, be

capable of exhibiting the changes in entropy on

extension required to produce an entropic elastic

force. (A) Space-filling representation showing all of

the Rieske Iron Protein in gray, but allowing only

one tether to be seen. (B) A similar view to that in

A, but using the ribbon representation that allows

both tethers to be seen in symmetric relation in an

open region of the structure. (Prepared using the

crystallographic results of Lange and Hunte^^ as

obtained from the Protein Data Bank, Structure File,

IKYO.)

8.3 The Electron Transport Chain: Protein Machines as Redox-driven Proton Pumps

381

Heme b

Rieske ->

Fe-S Center

Heme b

Rieske

Fe-S Cen

FIGURE

8.19.

Stereo views of monomers of Complex

III:

cytochrome bci complex of chicken with (A) and

without (B) inhibitors. Protein in backbone repre-

sentation with neutral residues in hght gray,

hydrophobics in gray, aromatics in black, and

charged residues in

white.

(A) The tether is extended

when the FeS is at Qo site. (Prepared using the crys-

tallographic results of Zhang et

al.^^

as obtained from

the Protein Data Bank, Structure File 3BCC.) (B)

The tether is relaxed when the FeS center is at the

heme

site. Note the extension of the tether that is

apparent by observing the distance between

A66

and

174.

(Prepared using the crystallographic results of

Zhang et al.^^ as obtained from the Protein Data

Bank, Structure File IBCC.)

loaded?"The foregoing structural analysis does

indeed indicate that hydrophobic association

spring-loads the "iron-sulfur protein of the bci

complex." Now, if this is to provide a meaning-

ful step forward in understanding the mecha-

nism, an explanation must follow whereby the

favorable hydrophobic association involving

the ubiquinol at the Qo site becomes lost so that

the elastic retraction can bring about domain

movement. The explanation comes from the

change in the Gibbs free energy of hydropho-

bic association due to the contribution of

apolar-polar repulsive free energy of hydration

(AGap) that arises from charge formation.

382

8. Consilient Mechanisms for Protein-based Machines of Biology

8.3.4.3.6 Formation of QHf Would Disrupt

Hydrophobic Association at the Qo Site Due

to AGap

Perhaps the most fundamental and certainly

the most dramatic observation during the study

of hydrophobic association using the elastic-

contractile model proteins has been that the

introduction of charge disrupts hydrophobic

association. This finding and indeed the

principal basis for controlling hydrophobic

association, AGap, stands out in the release of

hydrophobic association at the Qo site. The

transfer from ubiquinol, QH2, of one electron

to the FeS center and of a second electron to

the heme bL center leaves a positively charged

ubiquinol, for example, QH2^.

Before electron transfer, as fluctuations

toward dissociation occur with occupancy by

the hydrophobic QH2 molecule, sufficient

hydrophobic hydration would form to result in

water insolubility such that reassociation

results. If, on the other hand, fluctuations

toward dissociation occur for QHf^ occupancy,

as hydrophobic hydration formed it would be

recruited to align for hydration of the positively

charged QH^^. Thus, increased hydrophobic

hydration that ensured hydrophobic reassocia-

tion would not occur and hydrophobic dissoci-

ation would be the result. Without the favorable

decrease in Gibbs free energy for association,

the deforming force that caused extension of

the tether would be gone, and elastic retraction

would result. Additional mechanical details of

the resulting domain movement follow below.

8.3.4.3.7

Mechanics Whereby the Stretched

Tether Could Elastically Contract to Relocate

the FeS Center from the Qo Site to the

Cytochrome Ci Heme Site

The expected mechanics for relocation of the

FeS center within the globular domain of the

RIP come from the yeast Complex III depicted

in Figure 8.17. One of the two RIP subunits

occurs in backbone representation such that it

is possible to see through to the underlying Qo

site for hydrophobic association. Immediately

below the extended tether resides a raised side

chain of residue F169. The bulky F169 side

chain is positioned to function as a fulcrum that

bears the force of the elastic contraction. This

raises the stylus-like point of the FeS center

(see Figure

8.16B)

out of the pit of the Qo site

and over the L262 and V264 rim of the pit sep-

arating the Qo and heme Ci sites. In this manner

the elastic contraction would lift the globular

domain and rotate it into the less hydrophobic

heme Ci site for electron transfer. Transfer of

the electron to the heme Ci and replacement

at the Qo site of a hydrophobic ubiquinol

molecule re-establishes the favorable Gibbs

free energy for hydrophobic association at the

Qo site and positions the FeS center for the next

electron transfer.

8.3.4.3.8

Proposed Calculation of Tether

Dynamics to Assess Development of Entropic

Elastic Force on Observed Extension

The observed structural changes attending the

movement of the globular domain of the RIP

are exactly as one would expect for the

hydrophobic and elastic consiHent mechanisms.

Nonetheless, one should make and repeatedly

test predictions based on the proposed mecha-

nism, as was done over and over again when

establishing the consilient mechanism for the

diverse energy conversions of the designed

elastic-contractile model proteins (see Chapter

5).

In this way a proposed mechanism can

achieve the required evaluation for a given

system. The most meaningful tests are those for

which there is a physical result obtained from

instrumentation, not a result from a "computa-

tional experiment." Our ultimate goal is, of

course, to reach the stage where the under-

standing of the forces involved is such that

suf-

ficiently reliable mathematical descriptions can

be formulated. The present state of such calcu-

lations is not yet at that stage. In our view, using

a low dielectric constant to make ion pairing

favorable instead of an apolar-polar repulsive

free energy of hydration demonstrates the

present limitations. Even so, the ultimate goal

remains. Accordingly, with extensive data on

the versatile elastic-contractile model proteins

of this book, a computational approach that

served the model system well could become a

meaningful step with which to clarify the mech-

anism of interest.

8.3 The Electron Transport Chain: Protein Machines as Redox-driven Proton Pumps

383

One computational test would be to perform

a molecular dynamics calculation of the change

in internal chain dynamics of the observed

extension, that is, to calculate the change in

chain entropy resulting from the extension

observed in Figure 8.19. The two available

tether sequences are residues 64 through 74 (A

DVLAMSKIEI)of the chicken sequence

and residues 84 through 94(ADVLAMAK

V E V) of the yeast sequence.

8,3.4.4 Consilient Mechanism for Proton

Transport at Qo and Qt Sites

The focus has been on both aspects of the con-

silient mechanism (hydrophobic association/

dissociation and elastic force development/

relaxation) involved in the unique domain

movement for electron transfer within

Complex III. In what follows, the same aspect

of hydrophobic association/dissociation of the

consilient mechanism is proposed for facilitat-

ing proton gating.

TTie scenario often repeated throughout

this book speaks of associating hydropho-

bic domains in which fluctuations in the direc-

tion of dissociation proceed until so much

hydrophobic hydration has developed that

hydrophobic association (insolubility) recurs.

However, if a charged group should appear

suf-

ficiently proximal to the hydrophobically asso-

ciating domains, now as the fluctuation towards

dissociation occurs, the hydration that enters

is immediately oriented for hydration of the

charged group such that reassociation does not

occur, because insufficient hydrophobic hydra-

tion formed to result in insolubility. Accord-

ingly, charge disrupts hydrophobic association

by disrupting hydrophobic hydration. This com-

petition for hydration that blocks the build up

of too much hydrophobic hydration can often

be easily quantified. In many cases a change in

hydrophobicity results in a very large change in

pKa or in the affinity for other counterions.

In such cases the change in Gibbs free energy

for apolar-polar repulsion, AGap, becomes

easily quantified. For example, AGap = 2.3 RT

ApKa (see Equation [5.13]).

In the context of proton gating, at both the

Qo and Qi sites charge formation occurs as the

result of electron transfer. Herein lies the cou-

pling of electron transport to proton pumping.

By means of AGap, the formation of charge

opens aqueous access for proton egress and

ingress. As shown in Figure 8.20, both sites

exhibit "waters of Thales" that constitute

incipient water channels through which

protons could possibly move. The presence

of hydrophobic groups, however, with the

apolar-polar repulsive free energy of hydra-

tion, require that a greater aqueous pathways

be formed, as considered below.

8.3.4.4.1

Proton Access to Cytosol from

the Qo Site

It has already been argued above that forma-

tion of the positively charged QH2^ triggers

the displacement of the globular domain of the

RIP from the Qo site. Formation of the posi-

tive charge on electron transfer from QH2

disrupts the hydrophobic association of a very

hydrophobic tip of RIP with a substantially

hydrophobic pit of the Qo site. This is shown

in Figures 8.15, 8.16, and 8.17. By this element

of the consilient mechanism, competition for

hydration between hydrophobic and charged

residues gives rise to positive cooperativity,

wherein the appearance of charge at one loca-

tion facilitates charge formation, for example

by ion-pair separation, at another location.

Accordingly, charged groups cooperate to

destructure hydrophobic hydration and

thereby propagate an environment for charged

groups.

Based on studies of the elastic-contractile

model proteins reviewed in Chapter 5, during

contraction by hydrophobic association proton

diffuses in the contractile matrix at the rate of

one-fifth that of the diffusion of water out of

the matrix. Specifically, 20 Mrad y-irradiation

cross-linked poly(GVGVP), that is, X^^-

poly(GVGVP), contracted by hydrophobic

association and lifted a weight more than five

times faster on addition of 0.15 N NaCl and

0.01

M sodium phosphate (PBS) at pH 7.4

and 25°C'^ than did X'°-poly[4(GVGVP),

(GVGVP) in PBS on lowering the pH from 7.4

to 2.1 at 37°C.^^ Despite there being more

than 60% water in the matrix of the elastic-

384

8. Consilient Mechanisms for Protein-based Machines of Biology

Cytoplasmic^

side .> r*^

lipid layer

of inner

mitochondrial

membrane

Matrix side iJ^

H^ne

erne Ci

site

erne

FIGURE 8.20. Yeast cytochrome bci stereo view of

hemes, the ubiquinol Qo site and the ubiquinone Qi

site,

and the "waters of

Thales."

AGap,

the devel-

opment of a positive charge at the Qo site on oxida-

tion of ubiquinol disrupts the otherwise most

favorable hydrophobic association of the FeS center

of Reiske Iron Protein and allows for elastic retrac-

tion by the extended tethers. Removal of the FeS

center of the Reiske Iron Protein from the Qo site

allows release of the protons of QH2^^ to the cyto-

plasmic side of the inner mitochondrial membrane.

Furthermore, development of a negative charge at

the Qi site on reduction of ubiquinone to form Q"^

opens an aqueous channel for the uptake of two

protons from the matrix side to reconstitute

ubiquinol,

QH2.

(Prepared using the crystallographic

results of Hunte et al.^^ as obtained from the Protein

Data Bank, Structure File, lEZV.)

contractile model protein, we interpret this to

mean that water inside of the matrix is sub-

stantially hydrophobic hydration, which is

unsuited for hydration of proton. Stretch-

induced and hydrophobic-induced pKa shifts

provide the same argument that hydrophobic

hydration is unsuited for hydration of charged

species (see Chapter 5).

8.3.4.4.2

AGap Control of Proton Ingress from

the Matrix to the Qi Site

As shown in Figure 8.20, the Qi site, even before

the presence of negatively charged ubiquinone,

exhibits strings of water molecules emanating

toward the matrix side of the inner mitochon-

drial membrane. As stated by Hunte when dis-

cussing associated ionizable residues of H181

and E272 of RIP, "These residues are connected

with the bulk solvent, either directly or by a

short array of hydrogen-bonded, buried water

molecules.. ."^"^ Adding to these potential

charges, reduction of ubiquinone provides for

the formation of as many as two additional

negative charges, that is,

Q^~.

By means of

AGap,

association of proximal hydrophobic groups

would be disrupted on Q^" formation. This

would have the effect of expanding the strings

of water molecules into aqueous channels

capable of allowing the influx of protons at

proper rates from the matrix to complete the

conversion of ubiquinone to ubiquinol and

thereby to couple electron transfer within the

lipid layer of the inner mitochondrial mem-