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

365

8.3.2.3.2

Proton Release to the Cytoplasmic

Side of the Inner Mitochondrial Membrane

As redox systems are not known to be near the

cytoplasmic side of the inner mitochondrial

membrane, it is more difficult to consider oxi-

dation sites where formation of the positively

charged species, QH^^ and FMNH2^, could

occur. These are both species that, theoretically,

could release protons to the cytoplasmic side of

the membrane to complete proton transit in a

tightly coupled way across the inner mitochon-

drial membrane. These positively charged

species are expected to exhibit decreased

lipid solubility, although the intramolecular

ion pairing of negatively charged phosphate to

the positively charged isoalloxazine ring of

FMNH2^ might improve the possibility of FMN

use.

Obviously, our limited knowledge of the

molecular structure of Complex I does not

allow sufficient insight to make a specific

consihent-based proposal.

83.2.4 Dependence of Reduction Potential

on Hydrophobicity of Binding Site?

As was shown for N-methyl nicotinamide in

Figure 5.20C, the reduction potential of nicoti-

namide varies with hydrophobicity of binding

site.

This allows the possibility that ubiquinol

could be oxidized at two different sites at the

cytoplasmic side of the inner mitochondrial

membrane, but the question becomes oxidized

by what, as the redox centers have been con-

sidered to be on the matrix side of the mem-

brane. These problems with understanding

proton transport by Complex I are not present

with Complex III, as discussed in section 8.3.4.

8.3.3 Complex II:

Succinateiubiquinone Reductase—

Reduction of FAD by Succinate to

Produce FADH2, Which Ultimately

Results in Reduction of Ubiquinone

to Produce Ubiquinol for Subsequent

Reactions of the Electron

Transport Chain

Mammalian Complex II contains four subunits.

The larger two subunits are extramembrane on

the matrix side. They are a 79kDa flavoprotein

and a 29kDa iron-sulfur protein with three

iron sulfur centers: Fe2S2, Fe4S4, and Fe3S4. The

two membrane bound subunits bind a single

heme b and are reported to contain two or

more ubiquinone/ubiquinol binding sites.

8.3.3.1 Function of Succinate

Dehydrogenase, the E. coli Analogue of

Complex II (Succinate.'ubiquinone

Reductase)

The Singular Purpose of Succinate Dehydroge-

nase is to Provide Reduced Ubiquinone, That

is,

Ubiquinol, to the Inner Mitochondrial Mem-

brane as an Electron Carrier and as Chemical

Energy for Proton Transport. The overall reac-

tion is as follows:

OOCCH2CH2COO + Q = OOCCH =

CHCOO +QH2

Succinate + Ubiquinone =

Fumarate + Ubiquinol (8.12)

Equation (8.9) is a reversible reaction; it can

run either direction by changes in the relative

concentrations of succinate and fumarate. Even

so,

biology has different enzymes specializing in

producing fumarate from succinate under

aerobic conditions and for producing succinate

from fumarate under anaerobic conditions.

The two enzymes appear essentially identical

with the same prosthetic groups, that is, FAD

and the same three iron sulfur centers. There

is,

however, an important difference of side

products of reactive oxygen species (ROS)

that is greater in the reaction catalyzed by

fumarate reductase enzyme (see section

8.3.3.4

below) than in the reaction catalyzed by

succinate:ubiquinone reductase. It has to do

with a small difference in the accessibility of the

flavin prosthetic group to the aqueous phase.

The flavin appears better shielded from dis-

solved oxygen in succinate:ubiquinone reduc-

tase.^"^

Obviously, the electron transport chain

of the inner membrane of the mitochondrion

must function under aerobic conditions as the

terminal reaction due to Complex IV

(cytochrome c reductase) involves molecular

oxygen (O2) as a reactant, that is, the reduction

of oxygen to water.

366

8. Consilient Mechanisms for Protein-based Machines of Biology

83,3,2 Succinatembiquinone Reductase in

Three Distinct Reaction Steps

8.3.3.2.1

Step 1: Succinate Reduction of FAD

to Produce FADH2 and Fumarate

OOCCH2CH2COO +FAD

OOCCH

CHCOO

+ FADH2

(8.12a)

In this reaction, the protons and electrons

transfer together as hydrogen atoms.

8.3.3.2.2

Step 2: Oxidation of FADH2 to FAD

by Transfer of Electrons to FeS Centers with

Release of Protons to Matrix

FADH2

+ 2FeS =

FAD

+ 2FeS +

IW (8.12b)

This reaction of Complex II begins the separa-

tion of electrons from protons. However, why

does Complex II not contribute to proton

transport? If

FADH2

were oxidized on the cyto-

plasmic side of the lipid bilayer membrane, then

conceivably the two protons could be released

to the cytosol, as required for effective proton

transport.

8.3.3.2.3

Step

Transfer of Electrons to

Ubiquininone (Coenzyme Q) from FeS

Centers to Produce Negatively Charged

Ubiquinone,

Q^~,

Which on Receipt of Protons

from Matrix Produces QH2

2FeS

+ Q + 2H

2FeS + QH2

(8.12c)

The reduction of ubiquinone by receipt of two

electrons to produce

Q^"

within the lipid bilayer

membrane, but yet on the matrix side, would

allow the pick up of two protons from the

matrix. The process would result from forma-

tion of the negatively charged Q^~ whereby

the competition for hydration due to the

apolar-polar repulsive free energy of hydra-

tion,

AGap,

would open an aqueous channel to

the matrix side of the membrane for entry of

two protons, 2H^. With the above-preferred

location for reaction (8.9b) combined with the

just noted location of reaction (8.9c), there

would be a net transport of two protons from

the matrix side to the cytoplasmic side of the

membrane. This would be in keeping with the

direction of proton transport for Complexes I,

III,

and

IV.

As shown below in Figures 8.8 and

8.9, however, transport could not occur by this

utilization of hypothesis 1 of the consilient

mechanism for proton pumping (see section

8.3.1.2), because the arrangement of the redox

centers is not as required to release two protons

to the cytosol. In keeping with this perspective,

of course. Complex II does not pump protons.

Nonetheless, when ubiquinol is used by

Complex III, recall that the two protons that

added to ubiquinone to form ubiquinol came

from the matrix side.

8.3,3,3 Structure of Succinate

Dehydrogenase, the E. coli Analogue of

Complex II (Succinate-ubiquinone

Reductase)

The molecular structure of the analogue for

Complex II, succinate dehydrogenase from E.

coli, is shown in Figure 8.8^"^ to exhibit the

general form shown in Figure 8.6. Figure 8.8A

uses a space-filling representation where

neutral residues are light gray, aromatic

residues are black, other hydrophobic residues

are gray, and charged residues are white. With

this means of representation, immediately

apparent is a hydrophobic tip for insertion into

the lipid layer of the membrane. Also apparent

is a larger globular, more hydrophilic com-

ponent that resides within the matrix side of

the membrane.

With the ribbon representation in Figure

8.8B, a more transparent view of the structure

allows visualization of the redox groups and the

distribution of water molecules, shown as light

dots.

The FAD resides nearly centered within

the globular component and is associated with

very few water molecules. Then, in a stepwise

manner, the three iron-sulfide centers fill in

between FAD and the lipid bilayer, and just

within the region of the lipid bilayer resides the

heme b group. Reduction of ubiquinol occurs

between the Fe3S4 center. On removal of the

protein, this distribution of redox centers

becomes more apparent in Figure 8.9A,B.

An expanded view of the FAD group is

shown in Figure 8.9C along with the inhibitor,

oxaloacetate, that resides in the substrate (suc-

cinate) site. FAD is shown to be the phosphate

to phosphate coupling of adenosine monophos-

phate (AMP) with FMN. The key point of

Figure 8.9C is the proximity of substrate to the

!.3 The Electron Transport Chain: Protein Machines as Redox-driven Proton Pumps

Cytoplasmic side

Insertion into lipid

(biydrophobic) layer

of membrane

367

Matrix side

Heme

Insertion into lipid

(hydrophobic) layer

of membrane

*- Fe3S4

VFe4S4

"^FejSj

-•-FAD

FIGURE 8.8. Stereo view of E. coli succinate:

ubiquinone oxidoreductase, which is an analogue of

Complex II of the electron transport chain of mito-

chondria, with neutral residues light gray, aromatics

black, other hydrophobics

gray,

and charged residues

white. (A) Space-filling representation with water

three-ring aromatic redox group isoalloxazine

of the flavin component. This shows the reduc-

tion of FAD to form FADH2 to occur by direct

transfer of the two hydrogen atoms, that is,

without physical separation of proton from

electron. Thus there is no opportunity for for-

mation of an anion, such as FAD^", that could

then pick up two protons from the matrix side

of the membrane as one part of the two-part

process of proton transport from one side to the

other of the lipid bilayer membrane.

shown as light dots. (B) Ribbon representation with

ligands and "waters of Thales." (Prepared using the

crystallographic results of Yankovskaya et al.^"* as

obtained from the Protein Data Bank, Structure File,

INEN.)

Reduction of ubiquinone by addition of two

electrons on the matrix side of the membrane

could be the basis for the pick up of two protons

from the matrix side. However, the second part

of the release of two protons to the cytoplasmic

side of the membrane to achieve net transport

of two protons is absent. Thus, from a structural

standpoint, it is understandable that Complex

II does not contribute to proton pumping.

Complex II does make an important contribu-

tion of diffusible ubiquinol to the membrane

368

8. Consilient Mechanisms for Protein-based Machines of Biology

Ubiquinone site

fl*r^*-FAD

.$*^^J^^

Isoalloxazine

ring

|-«-

FMN -•I

FIGURE 8.9. Stereo view of E. coli succinate:

ubiquinone oxidoreductase, an analogue of Complex

II of the electron transport chain of mitochondria.

(A) Prosthetic groups (ligands) and "waters of

Thales."

(B) Ligands

(FAD,

FeS

centers,

heme

etc.)

that becomes oxidized by Complex III as part

of the quinone cycle of proton pumping as part

of electron transport.

8.33.4

Fumarate Reductase from

Wolinella succinogenes, Another Analogue

for Complex II of the Electron Transport

Chain of Mitochondria

Before structural determination of succinate

dehydrogenase, fumarate reductase provided

f substrate

site

reaction site. (Prepared using the crystallographic

results of Yankovskaya et al.^'^as obtained from the

Protein Data Bank, Structure File, INEN.)

insight into the structure and function of

Complex II (succinate.ubiquinone reductase) of

the electron transport chain."^^ As noted above,

the succinate dehydrogenase reaction (8.9) suc-

cinate + ubiquinone = fumarate + ubiquinol, is

reversible, and in Wolinella succinogenes the

function of fumarate reductase is the reverse, to

produce succinate from fumarate.^^

Figures 8.10 and 8.11 present the structure of

fumarate reductase in the same manner as used

in Figures 8.8 and 8.9 for succinate dehydroge-

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

369

nase.

Figure 8.10A shows in stereo view and

space-filling representation the dimeric state of

fumarate reductase with neutral amino acid

residues in light gray, with aromatic residues in

black, other hydrophobic residues in gray, and

charged residues in white. Again, a hydropho-

bic band identifies the location of the lipid

bilayer membrane. Figure 8.10B uses the

ribbon representation of the protein, which

allows easy visualization of the a-helices and p-

sheet structural elements of the protein-based

machine, as well as of some of the distribution

of detectable water molecules.

Figure 8.11 A gives the redox groups in ball

and stick representation and the substantial

number of detectable "waters of Thales" as

Cytoplasmic side

Hydropliobic band

to reside in

lipid membrane

Matrix side

Hydrophobic band

to reside in

lipid membrane

FIGURE 8.10. Stereo view of

Wolinella succinogenes

fumarate reductase dimer, an analogue of succi-

nateiubiquinone oxidoreductase (Complex II) of the

electron transport chain of mitochondria, with

neutral residues light gray, aromatic residues black,

other hydrophobic residues gray, and charged

residues white. (A) Space-filling representation with

water shown as Hght

dots.

(B) Ribbon representation

with ligands and "waters of

Thales."

(Prepared using

the crystallographic results of Lancaster et al.^^ as

obtained from the.Protein Data Bank, Structure File,

IQLB.)

370

8. Consilient Mechanisms for Protein-based Machines of Biology

^-Heme b

Ubiquinone

site-*

Fe3S4

Fe4S4'

FezSj

FAD-«

- fe^ <ir

-* SB ii)

J^^^ ^^^IK-Heme

(J?

& ^

^"W**^ B^l^

^ • substrate

site

FIGURE 8.11. Stereo view of

Wolinella succinogenes

fumarate reductase dimer, an analogue of mitoc-

hondrial succinate :ubiquinone oxidoreductase

(Complex II) of the electron transport chain. (A)

Ligands in ball and stick representation and "waters

Thales"

shown as hght dots. (B) Ligands in space-

filling representation, showing close association of

substrate with the flavin isoalloxazine heteroaro-

matic three-ring system. (Prepared using the crystal-

lographic results of Lancaster et al.^^ as obtained

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

light dots while hiding the protein structure

itself.

In Figure 8.11B shown in space-filling

representation are the redox groups and sub-

strate and putative location of the ubiquinone

site in analogy to those of Figure 8.9 for succi-

nate dehydrogenase. In fact, the structural par-

allels between succinate dehydrogenase and

fumarate reductase with regard to distribution

of redox functions are essentially identical, but,

of course, in duplicate for the latter. Seemingly,

the only obvious difference in redox functions

is the presence of a second heme b in the latter.

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

371

This,

however,

without known functional con-

sequence, as both proteins seem to function in

the absence of the heme groups."^^'^^ Also, very

apparent is the greater number of detectable

water molecules proximal to the flavin group.

Although this organism functions anaerobi-

cally, molecular oxygen, when present, is an

order of magnitude more likely to become

ROS.^^

The disarming of ROS and their dam-

aging effects is the reason that antioxidants are

so beneficial. Of

course,

by the consilient mech-

anism, the action of ROS on proteins destroys

function, because such polar additions to pro-

teins limit the hydrophobic associations essen-

tial to function (see section 7.7 of Chapter 7).

Again, the structure provides an understand-

ing of why this protein with all of the redox

functions to effect proton transport does not

pump

protons.

As shown below, the structure of

Complex III (ubiquinonexytochrome c oxi-

doreductase) provides remarkable insight into

the coupling of electron flow to proton

pumping, especially when viewed from the

vantage point of the consilient mechanism with

its functional element of an apolar-polar repul-

sive free energy of hydration, AGap.

8.3.4 Complex III:

Ubiquinonexytochrome c

Oxidoreductase (The Cytochrome

bci Complex)

With the exciting achievement of molecular

structures for Complex ni,^"^'^^'^^^^ combined

with extensive studies on kinetics, thermody-

namics, and stoichiometry,"^^ came the first clear

insight into the coupling of electron transport

to transmembrane proton pumping. This pro-

cess,

so essential to sustain Life, is no longer

shrouded in mystery. The following constitutes

the first report whereby the proposed consiUent

mechanism extends our understanding of

mechanistic detail for the function of Complex

III,

and both hydrophobic and elastic consilient

mechanisms come into play.^^

The hydrophobic and elastic consilient mech-

anisms, two distinct but interlinked physical

processes of hydrophobic association/dissocia-

tion and elastic force development/relaxation,

couple to achieve movement. In functional

homodimer of Complex III, hydrophobic asso-

ciation of the RIP FeS center at the Qo site in

one monomer occurs with elastic force devel-

opment in the tether that connects the globular

component of the RIP to its membrane anchor

in the other monomer. Formation of the posi-

tively charged ubiquinol, as electrons pass to

the FeS center and to heme bL, effects

hydrophobic dissociation and allows relaxation

of the elastic forces whereby the extended

tether moves the FeS center to heme Ci for

transfer of its electron.

Proton gating represents another demonstra-

tion of the consilient mechanism in Complex

III.

The operative element of the change in

Gibbs free energy for hydrophobic association,

the apolar-polar repulsive free energy of

hydrophobic hydration,

AGap,

uses the develop-

ment of negative charge at the Qi site to open

an aqueous channel for uptake of proton from

the matrix side of the membrane. As noted

above, the formation of positive charge at the

Qo site displaces the hydrophobically associ-

ated RIP and enables release of two protons to

the cytoplasmic side of the membrane. At both

and Qi sites the formation of charge disrupts

hydrophobic association by means of

AGap

and

opens the door to proton egress and ingress.

These roles of the consihent mechanism are

demonstrated with structures of Complex III

reported in the literature and are available for

development and analysis from the Protein

Data Bank using programs such as FrontDoor

to Protein Explorer by Eric Martz, which is

available at no cost at www.proteinexplorer.

org.

8.3A.1 General Structural Description of

Complex III (Ubiquinol: cytochrome c

Oxidoreductase)

8.3.4.1.1

Composition

Eleven protein subunits comprise the monomer

of Complex III, but only three of these subunits

contain the redox

centers.

They are cytochrome

an integral membrane protein with two

hemes (bt and bn), the RIP on the cytoplasmic

side with a single a-helical membrane anchor,

and cytochrome Ci also located on the cyto-

plasmic side and also with a single a-hehcal

372

membrane anchor. Although the membrane

anchor for the RIP resides with cytochrome

b of one monomer, its FeS center interacts at

the Qo and cytochrome Ci sites of the other

monomer. Therefore, this is referred to as a

functional homodimer, wherein both mono-

mers are required to achieve function.

8. Consilient Mechanisms for Protein-based Machines of Biology

8.3.4.1.2

Structure

Complex III, shown in stereo view and in the

light-gray ribbon representation of Figure

8.12A,

exhibits twofold symmetry, as there are

two copies of each subunit related by a twofold

rotational symmetry axis perpendicular to the

•- Heme c

^ Heme Cj

membranS^^

lipid layer

cytosol

lipid layer

ofimier

mitochondrial

membrane

^After reduction

Rieske

FeS Center

Before reduction

site

Heme bg

matrix

FIGURE 8.12. (A) Complete stereo view of the

homodimeric structure (light gray) of yeast Complex

III,

cytochrome bci complex (ubiquinolxytochrome

c oxidoreductase), plus cytochrome c (gray at upper

right).

The location of the heme groups and the FeS

center are indicated in reference to the lipid bilayer

membrane. (B) Stereo view of redox centers showing

electron transfers, Q being for Coenzyme Q

Qj site

(ubiquinone), with Qo identifying the oxidation site

that becomes a positively charged site, for example,

QH2^^,

and Qi identifying the reduction site that

becomes a negatively charged site, that is, Q^". (Pre-

pared 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

373

membrane plane. Also shown in a darker gray

ribbon representation in Figure 8.12A is the

presence of a single molecule of cytochrome c

at the top righthand side in position to receive

an electron from the heme of cytochrome Ci

and to leave that site on the cytosolic side as the

reduction product of the overall reaction. The

prosthetic groups are shown again in stereo

view and in twofold symmetry in Figure 8.12B

plus the single cytochrome c heme center at the

upper right.

The ribbon representation shows transmem-

brane a-helices traversing through the lipid

bilayer membrane, containing heme b pros-

thetic groups, the Qo and Qi sites, and the FeS

center at the tip of the globular component of

the RIP and associated with the Qo site.

8.3,4.2 Phenomenology of Ubiquinol

Oxidation to Reduce Cytochrome c with

Net Proton Transport

8.3.4.2.1

Overall Reaction

The overall equation for the reaction catalyzed

by Complex III follows is given above in Equa-

tion

(8.7).

At first glance this balanced equation

seems to have redundant reactants and prod-

ucts.

Why does the equation have two QH2

molecules as reactant and yet give one QH2 as

product? Inversely for Q, why is there one Q

molecule as a reactant and two as a product?

Also,

why does the reaction read two protons

from the matrix with four protons being

released to the cytosol, and yet this reaction is

generally credited with pumping four protons?

Taking the last question first, the additional two

protons came from the matrix by means of

reduction of ubiquinone by Complex II. Thus,

by supplying ubiquinol to the membrane pool.

Complex II in reality contributes to the net

transport of four protons by Complex III.

The answer to the first two questions is that

Equation (8.7), in addition to stoichiometry,

reveals elements of mechanism. A bifurcation

of electron flow on oxidation of a single QH2

molecule at the Qo site occurs with the first

electron going to the FeS center and the second

passing through hemes be and be to add an

electron to Q at the Qi site. Now a second QH2

molecule at the Qo site repeats the bifurcation

to complete the reduction of ubiquinone at the

Qi site, which

Q^"

picks up two protons from the

matrix side of the membrane to complete

the stoichiometry of two QH2 molecules used

at the Qo site with one recovered at the Qi site.

The net result is two QH2 molecules used giving

two Q molecules, but one molecule of Q was

reduced, thereby regaining one molecule of

QH2.

8.3.4.2.2

Electron Transfer Steps

Focusing on the electron transfer steps indi-

cated in Figure 8.12B, a single QH2 molecule at

the Qo site passes one electron to the FeS

center; the FeS center then moves to the heme

Ci and reduces it; the reduced heme Ci transfers

its electron to the heme of cytochrome

c^^.

The

result is the production of one molecule of

cytochrome c^^ and QH2^ at the Qo site. The

QH2^

molecule at the Qo site then transfers a

second electron that passes through the b

hemes to ubiquinone at the Qi site. A second

QH2 molecule is similarly oxidized with the

reduction of a second cytochrome c^"^ to result

in two molecules of cytochrome c^^. Although

the electron transfer phenomenology is well

recognized and even the mechanism of electron

tunneling over significant distances is an

accepted mechanism, there remain issues of the

mechanism whereby domains associate/dissoci-

ate and move where and when necessary. These

points are discussed in terms of the hydropho-

bic and elastic consilient mechanisms, the prin-

cipal message of this book, in section 8.3.4.3.

8.3.4.2.3

Proton Transport Sites

Occupied Qo and Qi sites involved in proton

transport are shown in stereo view in associa-

tion with the heme bt and bn ligands in Figure

8.13A in ball and stick representation and in

Figure 8.13B in space-filling representation.

This is a monomer subset shown in stereo of the

Ugands that were shown in the homodimer of

Complex III in Figure 8.12B, which contained

in additional the heme Ci and c ligands. The

added schematic representation of Figure

8.13A illustrates that the oxidation due to the

removal of two electrons from a molecule of

ubiquinol at the Qo site sets the stage for trans-

fer of two protons to the cytoplasmic side of

the inner mitochondrial membrane. Also, the

374

8. Consilient Mechanisms for Protein-based Machines of Biology

Cytoplasmic side 21

sit^

FIGURE 8.13. Yeast cytochrome bci stereo view of

the membrane-bound Ugands—hemes bL and bn,

stigmatellin occupying the Qo (ubiquinol) site, and

ubiquinone at the Qi site. (A) Ball and stick repre-

sentation to show molecular backbone structures.

(B) Space-filling representation to give proper

volume representation. By means of

AGap,

ubiquinol,

on becoming positively charged by oxidation at the

site,

opens an aqueous channel to the cytoplasmic

side of the inner mitochondrial membrane for

proton release, and ubiquinone (coenzyme Q, CoQ),

on becoming negatively charged by reduction at the

Qi site, opens an aqueous channel for proton uptake

from the matrix side of the inner mitochondrial

membrane. (Prepared using the crystallographic

results of Hunte et

al.^^

as obtained from the Protein

Data Bank, Structure File, lEZV.)

reduction of a molecule of ubiquinone due to

the addition of two electrons results in the

uptake of two protons from the matrix side of

the inner mitochondrial membrane.

Ready visualization of this phenomenology

derives from the remarkable structural and bio-

chemical kinetic studies on Complex III. The

issue addressed below, however, becomes one

of understanding the intermolecular forces

whereby such proton transport is coupled to

electron transfer without significant leakage of

proton across the inner mitochondrial mem-