Lecture 25

The sources of energy: posphorylation, oxidation and coupling chemical energy to work

Peter Mitchell

Why protons?

Why ATP?

Why oxygen?

Most cells use a proton gradient as an energy source across their plasma membrane. Why do animal cells use a sodium gradient?

Why protons?

Protons because one single Histidine, Glutamate or Aspartate residue furnish a simple and tunable binding site for H+. pKa of these groups can vary by 2-3 units depending on the environment. These sites do not bind metal ions tightly unless work in concert.

It is much more difficult to build a selective binding site that would discriminate between Na+, K+, Mg2+, Ca2+, Zn2+, Cu2+, Pb2+, Hg2+, Fe2+, Fe3+, etc

Protomotive Force has an electrical component and can couple electrochemical proton gradient to the transport of other charged substances

mVinpHFH .....60

JoulesinFH

HRTH ...)

][

][ln(

2

1

-180 mV

pH = 8

0 mV

pH = 7

PMF = -60 -180 = -240 mV

Measuring the membrane potential…..

-180 mV

pH = 8

0 mV

pH = 7

Fluorescent dye Rhodamine 123 (Rh123+) will penetrate into the vesicles according to electric gradient, increasing their fluorescence. Increased concentration of Rh123 inside the vesicle beyond certain point will cause self-quenching.

positive charge

Calibration???Rhodamine 123

Measuring the membrane potential…..another way

-180 mV

pH = 8

0 mV

pH = 7

Valinomycin…potassium uniporter

K+

http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb1/part2/carriers.htm

What happens if we add valinomycin?

)][

][ln(

in

out

K

K

F

RT

but val. may affect the potential…

Measuring the membrane potential…..a better way

time

fluor

esce

nce

val

0.1

0.05

0.2

[KCl] outside mM

Null method

Why ATP?

The reaction of ATP hydrolysis is very favorable

ΔGo = -30.5 kJ/mol = - 7.3 kCal/mol

because:

1. Charge separation of closely packed phosphate groups provides electrostatic relief

2. Inorganic Pi, the product of the reaction, is immediately resonance-stabilized (electron density spreads equally to all oxygens)

3. ADP immediately ionizes giving H+ into a low [H+] environment (pH~7)

4. Both Pi and ADP are more favorably solvated by water than one ATP molecule.

ATP exists in complex with Mg2+

Mg2+

phosphoanhydride bonds

ATP is not the only:

Phosphoenolpyruvate (PEP) -61.9 kJ/mol

1,3-Bisphosphoglycerate -49.3 kJ/mol

Phosphocreatinine -43.0 kJ/mol

ATP -30.5 kJ/mol

Pyrophisphate (Pi-Pi) or

Inorganic polyphosphate (polyPi) -19 kJ/mol

Thioesters (Acetyl CoA) -31 kJ/mol

[ATP]

[ADP][P]lnRTGG o

p

Calculate Gp in erythrocytes if

Gp = -30.5 kJ/mol [ATP] = 2.3 mM;

[ADP] = 0.25; [Pi] = 1.65 mM,

In other cells: [ATP] [ADP] [AMP] [Pi] in mM

Rat myocyte 8 0.9 0.04 8.05Rat neuron 2.6 0.73 0.06 2.7E. coli 7.9 1.04 0.82 7.9

In real cells G for ATP hydrolysis is more negative than standard Go

ATP provides energy to group transfer reactions:

A-P → A + P G1

B-P → B + P G2

A-P + B → A + B-P G = G1-G2

ATP

1,3-BPGPEP

PEP

Phosphocreatinie

Glucose-6-P Glycerol-P

Pi

G o

f hy

drol

ysis

Synthesis of any phosphorylated compound can be coupled to ATP hydrolysis

Transfer reactions:Phoshoryl transfer; Pyrophosphoryl transfer; Adenylyl transfer

Synthesis of NTPs (dNTPs) from ATP occurs as phosphoryl exchange at G ~0. The reaction is catalyzed by nucleoside diphosphate kinase which first phosphorylates its own His, releases ADP and then phosporylates the incoming NDP or dNDP.

How is the energy of phosphoryl transfer (or removal) imparted to a conformational change or how the chemical work is done?

Simple binding of ATP to an enzyme (or another effector protein) may cause massive conformational change by allosteric mechanism through the stage of binding site rearrangement. Cleavage of the gamma phosphate would lead to another conformational change. A complete release of the nucleotide diphosphate returns the protein to its initial state.

Phosphorylation of certain sites (Tyr, Ser or Thr) promotes recognition by a counterpart domain (recall SH2 domains)

Why oxygen?

Electron re-distribution from less electronegative to more electronegative atoms occurs with massive energy release:

Electronegativity of common elements:

H < C < S < N < O …Cl < F

Identify the substance and the reduction state of the first carbon (red)

Enthalpies of oxidation (combustion)

hydrogen (MW 2)H2 + ½O2 → H2O -286 kJ/mol

methane (MW 16)CH4 + 3O2 → CO2 + 2H2O -891 kJ/mol

glucose (MW 180.2)C6H12O6 + 6O2 → 6CO2 + 6H2O -2840 kJ/mol

(-680 kCal/mol)palmitic acid (MW 256.4)C16H32O2 + 23O2 – 16CO2 + 16H2O - 9730 kJ/mol

Oxidation-reduction (Red-ox) reactions usually lead to re-distribution of electron densities or complete transfer of electrons resulting in change of ionization state.

Fe2+ + Cu2+ → Fe3+ + Cu+

or in the form of half-reactions: Fe2+ → Fe3+ + e

Cu2+ + e → Cu+

In biological systems oxidation is often coupled to dehydrogenation.

1. Direct transfer of electrons

2. As a transfer of H atoms or removal of H atoms coupled to production of H+

3. As a Hydride ion :H–

4. Through direct combination with oxygen

R-CH3 + (½)O2 → R-CH2-OH

Standard Reduction potentials for some half-reactions, Volt

½ O2 + 2H+ + 2e → H2O +0.816

Fe3+ + e → Fe2+ +0.771

Cytochrome c (Fe3+) + e → Cytochrome c (Fe2+) +0.254

Fumarate2- +2H+ + 2e → succinate2- +0.031

2H+ + 2e → H2 (standard condition) 0

Pyruvate + 2H+ + 2e → lactate -0.185

FAD + 2H+ + 2e → FADH2 -0.219

S + 2H+ + 2e → H2S -0.243

NAD+ + H+ + 2e → NADH -0.320

NADP+ + H+ + 2e → NADPH -0.324

α-ketoglutarate + CO2 + 2H+ + 2e → isocytrate -0.38

2H+ + 2e → H2 (pH 7) -0.414

Reduction potentials for mixtures of reductant/oxidant (hlf-reaction potentials) are measured using the standard hydrogen electrode

http://www.chemguide.co.uk/physical/redoxeqia/eomgdiag.gif

2H+ + 2e → H2 Mg2+ + 2e → Mg (metal)

donor][electron

acceptor][electron ln

nF

RTEE o

Electron transport chain puts reductants and oxidants in specific order

Complex I II III IV

Energy released in forming water is stored as a PMF

Energy is divided into smaller units ~12 protons per water molecule