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

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8.2 Overview of Energy Conversion in Biological Systems

355

adenine dinucleotide (FADH2, 2 molecules),

and adenosine triphosphate (ATP, 4 mole-

cules).

As ATP is the basic energy denomina-

tion of biology, the complete conversion of one

glucose molecule to the chemical energy cur-

rency of biology comes down to the oxidation

of NADH and FADH2 in the production of an

additional 32 molecules of ATP. This involves

five protein-based machines that constitute

what has been called oxidative phosphorylation

and that occur in the mitochondria, the so-

called energy factories of biology. Four of the

protein-based machines (Complexes I through

IV) effect the pumping of protons, and the fifth

complex utilizes the return of the protons

across the inner mitochondrial membrane to

produce the 32 molecules of ATP.

In general, then, the energy conversions of

biology reduce to the production of ATP and

the uses of ATP, that is, the production of

ATP by the five protein-based machines of the

inner mitochondrial membrane and the thou-

sands of subsequent protein-based machines

that do the necessary work of the cell. This

constitutes yet an enormous task that will fill

hundreds of volumes in the future of protein-

based machines. The intention of this volume,

however, is to add a simplifying feature of a

"common groundwork of explanation" for each

of the hydrophobic and elastic consilient

mechanisms. For the function of protein-based

machines of biology, this perspective recovers

an attractive element of simplification.

8.2,1.1 Photosynthesis for the Production

of Carbohydrate (Glucose)

The following statement of photosynthesis is

essentially a question of a balanced chemical

equation where each atom among the reactants,

on the lefthand side of the equation, is

accounted for in the products on the right-hand

side.

For photosynthesis.

Carbon Dioxide + Water + Light energy

6(C02) 6(H20)

^ Carbohydrate + Oxygen (8.4)

[C(H20)],

6(02)

The remarkable simpUcity of this statement,

known for over a century, is stunning. It

involves only the atoms of carbon (C), oxygen

(O),

and hydrogen (H) with truly simple stoi-

chiometry. While there is great beauty in the

simplicity of the equation, it obscures intricate

detail of just what does happen in the process

and, importantly, how it happens.

8.2.1.2 Respiration: Oxidation of Glucose

with Ultimate Reduction of Oxygen to

Result in Water and Carbon Dioxide

The chemical products of photosynthesis

become the chemical reactants of respiration,

and the chemical products of respiration

become the chemical reactants of photosyn-

thesis.

Of course, absent the light energy for pho-

tosynthesis, there would be no products of

carbohydrate and oxygen, and absent the chem-

ical reactants of respiration, there would be no

chemical energy with which to sustain

Life.

Thus,

instead of oxidizing glucose by fire to give rise

to heat,

occurs in fireplaces during the burning

of wood (which is cellulose composed of strings

of glucose molecules), living organisms evolved

the capacity, step by step, to turn the oxidation

of glucose into production of the energy con-

tained within the ATP molecule. For respiration.

Carbohydrate + Oxygen ^ Water

[C(H20)],

6(02) 6(H20)

+ Carbon Dioxide + Chem Energy

6(C02) (8.5)

Again, the simplicity of the chemically balanced

equation, this time representing respiration,

belies what may seem an almost bewildering

underlying set of complex reactions. Before

addressing the key energy-converting steps of

respiration, however, another pair of analytical

expressions points in the direction of recogniz-

ing the separation of the hydrogen atom into its

proton, ff, and its electron, e~, which represent

elemental features laid bare without the blur of

molecular detail.

Respiration can be seen as a pair of

half-

reactions, which provides initial insight into the

underlying process carried out by the metaboUc

machinery of the cell. For oxidation of glucose,

the oxidative half reaction is

C6H12O6 + 6(H20) ^ 6(C02) + 24H^ + 24e-

(8.6)

356

8. Consilient Mechanisms for Protein-based Machines of Biology

For reduction of oxygen, the reductive half

reaction is

6(02) + 24H^ +24e- ^ 12(H20) (8.7)

Addition of the two half-reactions gives the

expression for respiration on explicitly in-

cluding the statement for the chemical energy

obtained. The analytical simplicity of the

half-

reactions lays out the underlying essential result

of the biological electron transport chain.

Indeed, the electron transport chain of the mito-

chondrion achieves the separation into protons,

¥t, and electrons, e~. (For the structure of the

mitochondrion, refer to Figure 8.5, below.)

The protons are released to one side of an

otherwise generally proton-impermeable inner

mitochondrial membrane to collect the protons

in the space between the inner and outer

membranes of the mitochondrion. The resulting

proton concentration gradient then drives for-

mation of ATP by the quintessential protein-

based machine, ATP synthase, as the protons

flow back through the inner mitochondrial

membrane by means of another special path

effecting proton permeability. Thus there are

two fundamental questions. The first is, how

does electron flow within the membrane

achieve unidirectional proton flow across the

membrane? The second is, how does the return

flow of protons result in the formation of ATP,

the energy coin of biology?

Incredibly, the marvelous work of many bio-

chemists, biophysicists, and crystallographers

has provided reaction stoichiometries, mecha-

nistic details, and structures. It is a beautiful

evolving story into which we would like to

introduce the reasoning of the consilient

mechanisms. As presented in Chapter 5, the

hydrophobic and elastic consilient mechanisms

developed from inverse temperature transi-

tions of hydrophobic association exhibited by

elastic-contractile model proteins functioning

as protein-based machines capable of inter-

converting the same energies interconverted by

living organisms. In particular, we introduce

the insight of an apolar-polar repulsion into

considerations of understanding molecular

mechanism. First to be considered, however, are

a number of intermediate molecular players

and processes in the early oxidation stages of

glucose in preparation for utilization of the

reduced molecules in the biological electron

transport chain.

8.2,1.3 Products of Glucose Oxidation by

the Reactions of Intermediary Metabolism

Despite what at initial consideration may

appear as maze-like detail, the many reactions

that accomphsh respiration, given as the oxida-

tion of glucose to CO2 and water, group as two

cycles tied together by a single reaction. The

first cycle is the glycolysis cycle that by way

of the transition reaction feeds into the citric

acid cycle, also called the Krebs cycle or the

tricarboxylic acid cycle. As briefly enumerated

below, the products of these reactions either are

or become convertible to the universal energy

currency of biology, ATP.

8.2.1.3.1

Glycolysis Cycle

By the glycolysis cycle, the oxidation of glu-

cose yields two molecules of pyruvate, two

molecules of reduced nicotinamide adenine

dinucleotide (2 NADH), and two molecules of

adenosine triphosphate (2 ATP).

8.2.1.3.2

Transition Reaction

In the transition reaction, the two pyruvate

molecules are converted to two molecules of

acetyl coenzyme A, two molecules of carbon

dioxide (2 CO2), and two molecules of reduced

nicotinamide adenine dinucleotide (2 NADH).

8.2.1.3.3

Citric Acid (Krebs) Cycle

The Krebs cycle takes two molecules of acetyl

coenzyme A from the transition cycle and con-

verts them into four molecules of carbon

dioxide (4 CO2), six molecules of reduced

nicotinamide adenine dinucleotide (6 NADH),

two molecules of reduced flavin adenine dinu-

cleotide (2 FADH2), and two molecules of

adenosine triphosphate (2 ATP).

8.2.1.3.4 Summary of the Oxidation of

Glucose by Intermediary Metabolism

The products of intermediary metabolism for

the oxidation of one molecule of glucose are 6

molecules of carbon dioxide (6 CO2), 10 mole-

cules of reduced nicotinamide adenine dinu-

cleotide (10 NADH), 2 molecules of reduced

>.2 Overview of Energy Conversion in Biological Systems

A lev

js^mmmt'^r B

357

Fj particles

Mitochondrion

Fi particles

Inner

rDembrane

Cytoplasm (cytosol)

FIGURE

8.5.

The mitochondrion, the energy factory

the

cell.

(Top)

Electron micrographs

(A),

(B),

and (C)

of the inner mitochondrial membrane studded with

stalks and headpieces that are the extramembrane

components of ATP synthase with the remainder

contained within the inner membrane. (Bottom)

Drawing of

mitochondrion with an outer membrane

and a folded inner mitochondrial membrane enclos-

flavin adenine dinucleotide (2 FADH2), and

4 molecules of adenosine triphosphate (4 ATP).

8.2.2 Oxidative Phosphorylation in

the Mitochondrion: TTie Primary

Source of ATP

8.22,1

Structure

the Mitochondrion

A schematic representation of the mitochon-

drion and of vesicles derived from it, as well as

electron micrographs showing the stalk and

ing the matrix portion and separated from the outer

membrane by the cytoplasm, also referred to as the

cytosol. (A. Reprinted with permission from D.F.

AAAS.

and lower part reprinted from Biochem-

istry,

Second Edition,

Voet and

Voet, Copyright

1995,

John Wiley

Sons,New

York.

Reprinted with

permission of John Wiley &

Sons,

Inc.)

headpiece of ATP synthase extending from the

membrane, is given in Figure 8.5."^^ Further-

more, the four complexes of the electron

transport chain that produce the proton con-

centration gradient across the inner mitochon-

drial membrane and the fifth complex, ATP

synthase, that utilizes the proton concentration

gradient to produce ATP are schematically rep-

resented in Figure 8.6."^^

Part of our challenge, to assess the relevance

of the hydrophobic and elastic consilient

mechanisms and specifically of apolar-polar

358

8. Consilient Mechanisms for Protein-based Machines of Biology

Cytoplasmic

side

ADP

ATP

Complex Comolex Complex Complex Complex

FIGURE 8.6. Oxidative phosphorylation of the mito-

chondria, showing the four complexes of the electron

transport chain that pump protons across the inner

mitochondrial membrane into the cytoplasmic side

to produce a higher concentration of proton within

the inner membrane cytosolic space and showing the

ATP synthase (at right) that uses the proton flow,

repulsive free energy of hydration to oxidative

phosphorylation in the mitochondrion, resides

in the diversity of the structural entities

involved. In the abstract of an excellent and

timely review on free energy transduction of

mitochondrial respiratory enzymes, Schultz and

Chan wrote that "Each of the respiratory

enzymes uses a different strategy for perform-

ing proton pumping.'"^^

Of the four complexes involved in the

electron transport chain that achieve proton

pumping, crystal structures are available for

three. Fortunately, two known structures. Com-

plexes III and IV, pump two-thirds of the

protons that develop the proton concentration

gradient, as is much of the crystal structure

known for the fifth complex that produces the

ATP.

That complex, ATP synthase, utilizes the

proton concentration gradient to produce

resulting from the higher concentration within the

inner membrane space, from the cytoplasmic side to

the matrix side to produce ATP from ADP and Pi

(inorganic phosphate). (Reprinted with permission

from M. Saraste, "Oxidative Phosphorylation at the

fin de siecler

Science,

283,1488-1493,1999.^^ Copy-

almost 90% of the ATP obtained on oxidation

of glucose. Thus there exists an opportunity to

gain some perspective on the relevance of the

consilient mechanisms. Section 8.3 considers

Complexes III and IV in some detail, while

section 8.4 presents ATP synthase. First,

however, the overview and background per-

spectives continue.

8.2.2.2 Reactions of the Inner

Mitochondrial Membrane

The complete oxidation of one glucose mole-

cule to 6 molecules of carbon dioxide results in

the reduction of 10 NAD and 2 FAD to

produce 10 NADH and 2 FADH2 molecules.

Their oxidation by the inner mitochondrial

membrane results in the flow of electrons

through the four complexes of electron

8.2 Overview of Energy Conversion in Biological Systems

359

transport chain that pump protons into the

intermembrane space and reduces oxygen

molecules to water. The return of the protons

through the fifth complex, ATP synthase, results

in the production of 32 molecules of ATP. The

chemical energy obtained on oxidation of the

nucleotides, reduced on oxidation of one mole-

cule of glucose, ultimately appears in the for-

mation of 32 molecules of ATP.

This brings the total number of ATP mole-

cules formed from ADP and Pi to 36 in the

process of the oxidation of a single glucose

molecule. The set of reactions in the mitochon-

drion that produces 32 of the 36 ATP molecules

are collectively called oxidative phosphoryla-

tion.

This occurs in two steps. Step 1 utilizes a

series of four protein-based machines. Com-

plexes I, II, III, and IV, collectively referred to

as the electron transport chain.

ThQSQ

complexes

oxidize the 10 NADH and 2 FADH2 molecules

and pump protons across the inner mitochon-

drial membrane to produce a proton concen-

tration gradient. Step 2 involves the most

important energy converting protein-based

machine of biology (ATP synthase) that utilizes

the concentration gradient of protons across

the inner membrane of the mitochondria to

produce ATP from ADP and Pj.

8.2.2.3 Step 1: Oxidation of NADH and

FADH2, Via the Electron Transport Chain,

Pumps Protons into the Intermembrane

Space and Results in the Reduction of

Oxygen to Water

Step 1 involves four enzyme complexes with

reactions much as given by Schultz and Chan"*^:

Complex I: Oxidizes one molecule of NADH,

and, by an as yet unknown mechanism,

pumps 4 protons into the intermembrane

(cytosolic) space and reduces ubiquininone

(coenzyme Q) to produce ubiquinol, QH2.

Overall equation:

NADH

5H^ (matrix) + Q ^

NAD^

QH2 + 4H^ (cytosol) (8.8)

Complex II: FAD oxidizes succinate to pro-

duce fumarate and FADH2, which reduces

ubiquininone to produce ubiquinol, QH2.

Overall equation:

Succinate + Q ^ Fumarate

QH2 (8.9)

Complex III: The cytochrome bci complex

oxidizes 2 molecules of ubiquinol, pumps

4 protons into the intermembrane (cytosolic)

space, and reduces two molecules of

cytochrome c. Overall equation:

2QH2 + 2cyt c^^ + Q + 2H^ (matrix) -^

2Q-F2cyt c'-^ +QH2 +4HMcytosol) (8.10)

Complex IV: Cytochrome c oxidase oxidizes

cytochrome c pumps 2 to 4 protons into the

intermembrane (cytosolic) space and reduces

oxygen to water. Overall equation:

4cyt

c^-"

4H"'

(scalar) +

4H"'

(matrix) ->

4cyt c'^ + 2H2O

-h

4H^ (cytosol)

(8.11)

Complexes III and IV are considered in some

structural detail in section 8.3.

Based on the above outline of key elements

of the four complexes, 10 NADH molecules

would contribute about 100 protons and 2

FADH2 molecules would contribute another

12 protons. As indicated below, production of

32 ATPs by ATP synthase would utilize an

equivalent number, nominally 106 protons.

8.2.2.4 Step 2: Proton Driven

Phosphorylation Via ATP Synthase

The result of the oxidation of the reduced coen-

zyme molecules, 10 NADH and 2FADH2, is the

development of a proton concentration gradi-

ent across the inner mitochondrial membrane.

The return of the protons back across the inner

mitochondrial membrane results in the produc-

tion of 32 ATP molecules from 32 molecules of

ADP plus 32 molecules of Pi by means of the

ATP

synthase.

The understanding of this source

of energy for ATP synthesis derives from the

now well accepted, but once highly controver-

sial, Mitchell chemiosmotic hypothesis.^^

8.2.3 Question: Why Do Ions Form

and Why do Ions Pair?

Despite this issue having been addressed in

several contexts above, it is again stated sue-

360

8. Consilient Mechanisms for Protein-based Machines of Biology

cinctly here to emphasize the importance of the

perspective.

8.2.3.1 When There Is Adequate Water,

Ions Dissolve, That Is, They Prefer to Be

Fully Hydrated

Ions dissolve from salts, or whenever otherwise

ion paired, due to lowering of the ion

(self-

energy) free energy on achieving full hydration.

When there is inadequate hydration, the

driving force is for charge neutralization.

Neutralization of a negatively charged group

could occur by protonation or by pairing

with a cation, and neutralization of a positively

charged group would occur by loss of a proton

or by pairing with an anion. In either case the

neutralizing ion could be either an inorganic or

an organic ion, as in the case of the charged side

chains of amino acid residues.

8.2.3.2 AGap Provides the Driving Force

for Ion Pairing, Which Is an Expression of

Limited Availability of Water for

Hydration of the Ion

When a clustering of hydrophobic groups

occurs, there develops a competition for hydra-

tion between charged and hydrophobic groups

that raises the free energy of the charged species.

This results in an apolar-polar repulsive free

energy of hydration (AGap) that is seen as a shift

in pKa. Functional groups with a shift in pKa are

at an increase in free energy and would lower

that free energy by ion pairing. Ion pairing

within or between protein subunits is the result

of this AGap and is caused by the proximity of

hydrophobic groups. As concerns the protein-

based machine, it is the sequence-imposed coex-

istence of polar and apolar groups along a

protein chain that gives rise to association of

hydrophobic domains, and ion-pair formation

becomes an integral part of the energetics for

hydrophobic association. An apolar-polar

repulsive free energy of hydration also appUes

to oxidized and reduced states of prosthetic

groups and cofactors and to their interactions.

8.2.4 Question: Can Free Energy

Transduction Be Localized at Some

Crucial Part of the Enzymatic Cycle?

By the hydrophobic consilient mechanism of

energy transduction, the energizing of a protein

(an enzyme) results from AGap, the change in

apolar-polar repulsive free energy of hydration

due to molecules or inorganic ions interacting

with the enzyme or protein of interest.^^

Depending on the changes in the state of the

molecules or inorganic ions interacting with

the protein, the protein may even be energized

further, as on the splitting of ATP to form the

most energized state, ADP plus Pi. There then

occur stepwise decreases in the energized state

that generally results most dramatically on

release of Pi. The energized state abates as the

apolar-polar repulsions continue to relax. As

the cycle progresses, the enzyme becomes fully

de-energized only on return to its initial state.

Therefore, the answer to the posed question is

no;

the free energy transduction cannot be local-

ized at any particular part of the cycle, because

different amounts of energy reside within the

enzyme at different steps of the cycle, that

is,

due

primarily to apolar-polar repulsion, the ener-

gized state of the enzyme is different at each step

of the cycle.

8.2.5 A Glimpse of Selected Energy

Conversions Going Forward

This section provides in a relatively limited

detail a standard overview of energy conver-

sion. For all but the photosynthetic organisms,

however, energy conversion can be represented

in terms of three stages. The first stage is the

development of a proton concentration gradi-

ent across the inner membrane of the mito-

chondrion. The second stage is the use of that

concentration gradient to produce ATP, the

representative biological energy coin, again

centered on the inner membrane of the mito-

chondrion. The third stage is the use of ATP and

its equivalent nucleotide NTPs by ATPases or

in general by NTPases.

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

361

In subsequent sections one representative

protein-based machine is emphasized for each

stage. For proton pumping across the inner

mitochondrial membrane discussed in section

8.3,

Complex III (ubiquinonexytochrome c oxi-

doreductase), also called the cytochrome bci

complex, provides an amazing example of

both aspects of the consilient mechanism,

those controlling hydrophobic association, the

apolar-polar repulsive free energy of hydration

(AGap), and the development of elastic force

due to a decrease in internal chain dynamics

between two fixed points in a protein chain

segment. The singular choice of ATP synthase

also provides an opportunity in section 8.4 to

emphasize the use of AGap to energize ADP

plus Pi to form ATP. Among the myriad of

ATPases, the myosin II motor of muscle con-

traction, a linear contractile motor, discussed in

section 8.5 and as anticipated in Chapter 7, pro-

vides a remarkably apparent example of AGap,

whereby binding ATP disrupts hydrophobic

association of the amino-terminal domain with

the head of the lever arm, and loss of phosphate

re-establishes the hydrophobic association to

provide the powerstroke.

8.3 The Electron Transport

Chain: Protein Machines as

Redox-driven Proton Pumps

8.3.1 Introductory Comments

8.3.1.1 Structural Status of the Four

Protein Machines (Complexes 1,11, III,

and IV) of the Electron Transport Chain

8.3.1.1.1

Current Status of Structural Details

Available for the Four Complexes of the

Electron Transport Chain

Complex I is a very large protein machine with

about 43 subunits. Because of this the structural

information has only been achieved to date to

22 A resolution.^^'^^ The resolutions for Com-

plexes II (2.9 A), III (2.3 to 2.9 A), and IV (2.3

to 3.0 A) are nearly an order of magnitude

better and provide details at a level that allows

consideration of mechanism.

8.3.1.1.2

General Sources for More

Background Information

Two excellent reviews are available for the

protein-based machines of oxidative phospho-

rylation from which more background infor-

mation may be obtained. One is a clear and

succinct statement by Saraste,"^^ and the other,

by Schultz and Chan,"^^ is an excellent, more

extensive account of the state of information on

the structures, strategies, and thermodynamics

of the five protein-based machines of oxidative

phosphorylation. Both of these reviews may be

sought for additional insight and detail. In addi-

tion, the fifth edition of Biochemistry by Berg,

Tymozcko, and Stryer"*^ provides an outstanding

account of energy conversion in the mitochon-

drion. Our objective in section 8.3 is to consider

the structures and functions of the electron-

transport/proton-pumping complexes in terms

of the consilient mechanism. We begin by a

summary statement of simple hypotheses

whereby the apolar-polar repulsive free energy

of hydration, AGap, would play the pivotal role

of proton gatekeeper that is based on a struc-

tural analysis of Complex III and whereby

elastic force generation plays a pivotal role in

domain movement for selected electron trans-

fer. Due to both time constraints and limita-

tions in structural data, the other complexes are

not considered in as much detail.

8.3.1.2 Relevance of the Consilient

Mechanisms to the Coupling of Electron

Transport to Proton Gating/Pumping

8.3.1.2.1

Hypothesis 1: AGap as the

Gatekeeper for Transmembrane Proton

Pumping by the Electron Transport Chain

By the operative component,

AGap,

of the Gibbs

free energy of hydrophobic association, AGHA,

polar groups compete with hydrophobic groups

for hydration; the result is that the formation of

charged groups disrupts hydrophobic associa-

tion. Recognition of two aspects of the com-

petition becomes central to understanding

mechanism. One is that hydrophobic associa-

tion occurs whenever too much hydrophobic

hydration begins to form during an opening

fluctuation. Tlie second is the formation of a

362

8. Consilient Mechanisms for Protein-based Machines of Biology

charged group, whether positive or negative, that

recruits emergent hydrophobic hydration for its

own charged hydration and thereby effects

hydrophobic dissociation and the opening of an

aqueous channel.

Complex III, ubiquinonexytochrome c oxi-

doreductase, provides this example remarkably

well. Our proposal states that the oxidation of

ubiquinol, QH2, at the Qo site on the inter-

membrane side of the inner mitochondrial

membrane produces a positively charged

molecule, for example QH2^. Emergence of this

positively charged species due to the electron

flow of oxidation disrupts hydrophobic associ-

ation of the hydrophobic tip of the RIP with the

Qo site and thereby allows two protons, 2H^, to

pass into the intermembrane cytoplasmic space

with the result of a molecule of ubiquinone.

Furthermore, our proposal contends that the

reduction of ubiquinone at the Qi site produces

a negatively charged molecule, for example

Q^", on the matrix side of the inner mitochon-

drial membrane. Emergence of this negatively

charged species due to the electron flow of

reduction converts a string of hydrophobically

enclosed water molecules into a water-filled

channel for the entrance of two protons, 2H^,

from the matrix space to produce ubiquinol. In

this way by AGap, the formation of a positively

charged species opens the gate for proton

egress from the inner mitochondrial mem-

brane, and the formation of a negatively

charged species opens the gate for proton

ingress into the inner mitochondrial membrane.

Because of AGap and the structural locations of

the redox-formed positively charged and nega-

tively charged groups, the redox events and

proton transport events are tightly coupled.

Thus,

given the example of Complex III oxi-

dation of ubiquinol at the Qo site and reduction

of ubiquinone at the Qi site, we would like to

generalize the proton pumping mechanism to

the similar formation of a charged species of

whatever molecular basis at appropriate loca-

tions in the inner mitochondrial membrane.

General hypothesis of proton gating/pumping:

Proton translocation across the inner mitochon-

drial membrane occurs (1) by oxidative forma-

tion of

positively charged species that opens an

aqueous passageway to the cytoplasmic side of

the membrane and that becomes neutralized by

proton release to the intermembrane (cytoplas-

mic) space and (2) by the reductive formation of

a negatively charged group that opens an

aqueous passageway to the matrix side of the

membrane and that becomes neutralized by

proton uptake from the matrix space.

8.3.1.2.2

Hypothesis 2: Hydrophobic

Association Within Complex III of the

Hydrophobic (FeS) Tip of the Rieske Iron

Protein with the Hydrophobic Ubiquinol-

containing Qo Site Causes Extension and

Damping of Internal Chain Dynamics in the

Tether of the Iron Protein

Complex III exemplifies another aspect of the

consilient mechanisms. The FeS center of the

RIP resides at a very hydrophobic tip of a stylus-

hke structure with two sites for hydrophobic

association, one at the Qo site and a second at

the cytochrome Ci site. As part of our hypothe-

sis,

the relative affinity due to hydrophobic asso-

ciation at each site depends on the redox state

of the FeS center and the charge at the Qo site.

Before receipt of the electron, AGHA is more

favorable at the Qo site, whereas after receipt of

the electron and oxidation of ubiquinol, the

AGHA becomes sufficiently less favorable at the

Qo site to allow the elastic force of the stretched

tether to withdraw the FeS center from the Qo

site for translocation to the cytochrome Ci site.

On transfer of the electron to cytochrome

Ci,

the

affinity at this site decreases and the FeS center

returns to the very favorable hydrophobic

association at the Qo

site,

now occupied by a new

molecule of hydrophobic ubiquinol. The affinity

due to hydrophobic association is so favorable

at the ubiquinol-occupied Qo site that the

interconnecting chain segment between mem-

brane anchor and the globular component of the

RIP becomes stretched. We suggest that this is

yet another example of the elastic-contractile

mechanism demonstrated in the model proteins

discussed in Chapter 5 in the context of hydro-

phobic association causing extension of an inter-

connecting chain segment. Hydrophobic asso-

ciation coupled with damping of internal chain

dynamics on extension of interconnecting chain

segments results in development of elastic force.

Contraction by the muscle myosin II motor

is another example of hydrophobic association

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

363

on decreased charge, that is, on loss of phos-

phate, and one where damping of internal chain

motions on hydrophobic association must

surely exist as evidenced by an increase in the

elastomeric force under isometric conditions,

but the chain or chains involved have not been

as clearly identified.

8.3.2 Complex I: NADH:ubiquinone

Oxidoreductase—Oxidation of

NADH for Reduction of Ubiquinone

to Ubiquinol While Translocating Four

Protons from Matrix to Cytosol for

Every NADH Oxidized

8.3.2.1 Structural and Redox Center

Information for Complex I

The structure of Complex I of mammalian

mitochondria comprises an unusually large

complex of about 43 distinct protein subunits

with a three-dimensional structure yet to be

determined at atomic resolution. From electron

microscopy, it has been observed with an L-

shaped structure, as indicated in Figure 8.6 with

nm length in the membrane and an 8nm

protrusion into the matrix. Equation (8.5), the

overall reaction, NADH + 5 H^ (matrix) + Q ^

NAD^

+ QH2 + 4 H^ (cytosol), beUes a more

involved set of electron carriers. The process of

reducing ubiquinone, Q, to ubiquinol, QH2,

involves a substantial number of redox centers

with the coupled result of the transmembrane

transport of four protons for each NADH

molecule oxidized.

The process whereby oxidation of NADH

reduces ubiquinone to ubiquinol involves the

redox groups of flavin mononucleotide (FMN)

and six FeS centers. It appears, however, that the

membrane spanning subunits of the L-shaped

structure do not contain redox centers, but do

contain the ubiquinone. Images at 2.2 nm reso-

lution^^ demonstrate a structure more contorted

than the monolithic L-shaped block indicated

for Complex I in Figure 8.6. Accordingly, when

making suggestions of mechanism based on

AGap,

it is tempting to consider the possibility of

redox groups being sufficiently proximal to the

cytoplasmic side of the inner mitochondrial

membrane to form species such as QH^^ and the

analogous oxidized state of flavin, FMNH2^, to

provide a source of proton release to the cyto-

plasmic side of the inner mitochondrial mem-

brane. With regard to the matrix side of the

membrane, the possibility would continue with

the reduction of ubiquinone and FMN to form

negative species, such as Q^" and FMN^~, as

could happen on addition of two electrons to

each of the oxidized ubiquinone and flavin

rings,

that would open channels for protons to

enter from the matrix side of the membrane. Of

course, the problem with this analogy to the

quinol/quinone use of Complex III is the appar-

ent absence of structural information placing

flavin and ubiquinone appropriately at two sites

in the membrane component of Complex I.

8.3.2.2 Further Structural Considerations

8.3.2.2.1

Additional Detail for

L-shaped Structure

As shown in Figure 8.7 A for bovine Complex

I at 22 A resolution in ice, the monolithic block

of Figure 8.6 becomes a structure of a greater

defined shape with a bulbous matrix compo-

nent and a much more convoluted membrane

component. If the redox components, with the

exception of ubiquinone

itself,

reside in the

bulbous component, then the puzzle remains

one of how proton translocation occurs.

8.3.2.2.2

A re-arranged Horseshoe-shaped

Structure as a Candidate for the Native State

Further electron microscopic studies of an

enzymatically active Complex I show the

rearrangement from the L-shaped structure in

Figure 8.7B to the horseshoe-shaped structure

in Figure 8.7C.^^ This reversible rearrangement

due to a decrease in ionic strength reversibly

turned off (at high ionic strength) and turned

on (at low ionic strength). Specifically, raising

the concentration of NaCl to 0.2 M abolished

NADHidecylubiquinone reductase activity.

This appears to be a general salt effect on

hydrophobic association, as the same effect was

reported for KCl, LiCl, MgCl2, CaCl2, Li2S04,

and NaNOs, which is analogous to the results in

Figures 5.11 and 5.12 for elastic-contractile

model proteins. Accordingly, this gives the pos-

sibility that redox centers might occur within

the inner mitochondrial membrane.

364

8. Consilient Mechanisms for Protein-based Machines of Biology

Matrix side

FIGURE 8.7. Reconstructions from electron micro-

graphs of Complex I (NADH:ubiquinone oxidore-

ductase) of the electron transport chain of the inner

membrane of the mitochondrion. (A) A more

detailed L-shaped structure obtained at high ionic

strength. (B,C) Structures obtained at high ionic

8,3.2.3 Potential Prosthetic Groups for

Proton Uptake and for Proton Release

8.3.2.3.1

Proton Uptake from the Matrix into

the Inner Mitochondrial Membrane

The isoalloxazine ring of FMN is quinone-like

in that it can accept two electrons and take up

two protons on reduction and give off two

protons after oxidation that removes two

electrons. This becomes the mechanism for

proton translocation to be discussed below for

Complex III as integral to the ubiquinone

cycle. In particular, at the matrix side of the

Cytoplasmic side

strength (B) and at low ionic strength (C) for an

enzymatically active state. (Reprinted from Journal

of Molecular Biology, 277, N.

Grigorieff,

Three-

dimensional Structure of Bovine NADH:

Ubiqunone Oxidoreductase (Complex I) at 22A in

Ice.

membrane two electrons could be added to

ubiquinone to form Q^" and analogously to

FMN to give FMN^". By the apolar-polar repul-

sive free energy of hydration, AGap, of the

consilient mechanism, these charged species

transiently open channels for proton entry into

the membrane for neutralization of the charge.

One possible concern with using the isoallox-

azine ring is that it is a system of three fused

aromatic rings such that charge would not be as

localized as it is for the ubiquinone system or

for that matter for nicotinamide, both of which

can form single aromatic rings and take up

protons on reduction.