Biologic oxidations

Biological Oxidation

Involves the transfer of electrons: oxidation being termed for the removal of electrons & reduction for gain of electrons Oxidation is always accompanied by reduction of an e- acceptor Higher forms of lives completely rely on O2 for life processes i.e. respiration a process by which cells derive energy with a controlled reaction between H+ and O2; the end product being water.

However there do occur large no. of reactions in living system without the involvement of molecular O2. The reactions are catalyzed by a set of enzymes called as Dehydrogenases. Other reactions do incorporate molecular O2 for the completion of reaction. O2 is also required during treatment for respiratory and cardiac failure for, the proper functioning of both require O2.

Capturing energy from foods requires the transfer of electrons

Oxidation is the loss of electrons from one molecule or atom to another. Reduction is the gain of electrons by one molecule or atom from another.
Each intermediate compound in a biological oxidation is slightly more oxidized than its precursor

During a biological oxidation, energy is stored in the form of the chemical bonds in adenosine triphosphate (ATP). The energy produced when ATP is broken down to ADP and phosphate is used to power many processes in a cell.

Almost every chemical reaction in the cell either consumes or produces ATP at some point. The catabolic reactions in the cell are tightly coupled to the biosynthetic reactions, forming the two sides of metabolism: releasing energy by breaking things down and using energy to build things up.

The concept of the biological oxidation

 The three stages of

biological oxidation

10

MITOCHONDRIA
1.OUTER MEMBRANE MARKERS:

ACYL COA SYNTHETASE GLYCEROLPHOSPHATE ACYL TRANSFERASE 2.INTERMEMBRANE SPACE:

ADENYLATE KINASE
CREATINE KINASE 3..INNER MEMBRANE,OUTER MEMBRANE GLYCEROL-3-PHOSPHATE DH 4..INNER MEMBRANE INNER MEMBRANE: ELECTRON CARRIERS SUCCINATE DH

ATP SYNTHASE
MEMBRANE TRANSPORTERS 5.MITOCHONDRIAL MATRIX MARKERS: ~ENZYMES OF OXIDATIVE PROCESSES:INSIDE MITOCHONDRIA.

Enzymes involved in O/R reactions

Are k/as Oxidoreductases which includes : oxidases, dehydrogenases, hydroperoxidaes and oxygenases. Oxidases use oxygen as an electron acceptor Dehydrogenases cant use as an electron acceptor Hydroperoxidases use H2O2 as a substrate Oxygenases catalyse the direct transfer of O2 into the substrate Oxidases & dehydrogenases involved in respiration; hydroperoxidases neutralize free radicals & oxygenases are involved in biotransformation

Enzymes Involved in the Oxidation reactions

14

Catalyze the removal of hydrogen from a substrate with the involvement of oxygen as a H acceptor Exist in two different forms : some of them are copper containing as, Cytochrome oxidase - the terminal component of ETC which transfer the e - finally to O2. Other are flavoproteins as , L aminoacid oxidase, xanthine oxidase

Oxidases

Dehydrogenases
1.

Perform 2 main functions: Transfer hydrogen from one substrate to another in a coupled O/R reaction As components of Electron transport chain Dehydrogenases use coenzymes nicotinamides & riboflavin - as hydrogen carriers

2.

Hydroperoxidases
Includes 2 sets of enzymes : catalase and peroxidases Peroxidases reduce H2O2 at the expense of several other substances H2O2 + AH2 2H2O + A o Catalase uses H2O2 as electron acceptor & electron donor 2H2O2 2H2O Peroxisomes are rich in oxidases and catalases

Oxygenases
Catalyse the incorporation of O2 into subtrates in 2 steps - Oxygen is bound to the active site of the enzyme - bound O2 is reduced or transferred to the substrate Consists of two sets of enzymes 1. Dioxygenases : incorporate both atoms of oxygen into the substrate ; A + O2 AO2 2. Monooxygenases : incorporates one atom of oxygen into the substrate & the other is reduced to water A H + O2 + ZH2 A OH + H2O + Z

Mitochondrion oxidation

system

19

Electron Transport and Oxidative Phosphorylation

An Overview

Biological oxidations are catalyzed by intracellular enzymes. The purpose of oxidation is to obtain energy. Electron Transport: Electrons carried by reduced coenzymes (NADH or FADH2) are passed sequentially through a chain of proteins and coenzymes (so called electron transport chain)to O2 . Oxidative Phosphorylation: Coupling e- Transport (Oxidation) and ATP synthesis (Phosphorylation) . It all happens in mitochondrion or at the inner mitochondrial membrane (eukaryotic cells)

mitochondrion
the mitochondrion contained the enzymes responsible for electron transport and oxidative phosphorylation

In inner membrane knobs

Impermeable to ions and most other compounds

Oxidative phosphorylation is a metabolic pathway that uses energy released by the oxidation of nutrients to produce adenosine triphosphate (ATP) During oxidative phosphorylation, electrons are transferred from electron donors to electron acceptors (NAD/NADP+/FAD/FMN) In eukaryotes, these redox reactions are carried out by a series of protein complexes within mitochondria ,reactions are exergonic These linked sets of enzymes are called electron transport chains
COMPONENTS OF ETC;
EXCEPT COQ, ALL MEMBERS ARE PROTEINs
SOME HAS Fe-S CENTRES Fe RESIDUE COORDINATED WITH SH OF CYSTEINYL CYTOCHROMES HAS PORPHYRIN RING WITH Fe IN CENTRE CYT a+a3 HAS COPPER

Electron Carriers
The transfer of electrons is not directly to oxygen but through coenzymes There are 2 sites of entry NAD+ for electrons into the FMN electron transport chain: NAD+ or FAD
FeS FAD FeS ubiquinone Cyt b ubiquinone FeS Cyt c1 Cyt c Cyt a Cyt a3 1/2 O2

Both are coenzymes for dehydrogenase enzymes

Nicotinamide coenzymes: NAD+

X CONH2 XH2

H CONH2 + H+

N oxidised coenzyme + + NAD or NADP

N reduced coenzyme NADH or NADPH

Always a 2-electron reaction transferring 2 e- and 2 H+

The flavin coenzymes / flavoproteins

OH CH2 CH H3C N N N O
NH2 N OH CH2 CH H3C N N N O OH CH O OH CH CH2 O O P OH O O P OH O CH2 O N N N

OH CH O

OH CH CH2 O

O P OH OH

H3C

riboflavin monophosphate (flavin mononucleotide, FMN)

H3C

flavin adenine dinucleotide (FAD) OH

OH

FAD Always a 2-electron reaction transferring 2 e- and 2 H+

Oxidation and reduction of flavin coenzymes

O H C H3C H3C C C C H C C N CH2 HC HC HC H2C OH OH OH O O P OON C C N C NH C

O H C H3C
O H3C
-

O H C NH C H3C O H3C
-

H N C C C C N CH2 HC HC HC H2C OH OH

C C C

H N C C C C N CH2 HC HC HC OH OH

C NH C N H O

C C C H

C H

e +H

e +H

OH O O P OO-

OH O O P OO-

H2C

FMN

FMNH

FMNH2

it can accept/donate 1 or 2 e-. FMN has an important role in mediating e- transfer between carriers that transfer 2 e- (e.g., NADH) and those that transfer 1 e- (e.g., Fe+++).

Role of FMN mediating between 2e- & 1ecarriers: For example, when NADH donates electrons to the respiratory chain, the initial electron transfers are: NADH + H+ + FMN NAD+ + FMNH2 FMNH2 + Fe+++ FMNH + Fe++ + H+

Iron-sulfur Centers (clusters)

Iron-sulfur centers (Fe-S) are prosthetic groups containing 14 iron atoms Iron-sulfur centers transfer only one electron, even if they contain two or more iron atoms. E.g., a 4-Fe center might cycle between redox states: Fe+++3, Fe++1 (oxidized) + 1 e- Fe+++2, Fe++2 (reduced)

Cys S S Cys Cys S S Fe

S S Fe Fe S Fe

Fe S

S Cys Cys S S Cys

Fe S S Cys

Cys

Iron-Sulfur Centers

NAD+

Ubiquinone
O CH3O

FMN

Other names and abbreviations:

Coenzyme Q CoQ Q
FAD FeS

FeS ubiquinone Cyt b

CH 3 CH 3

CH3O O

ubiquinone FeS

(CH 2 CH

CH 2)nH

coenzyme Q

2 e- + 2 H+
OH CH3O CH 3 CH 3 CH3O OH (CH 2 CH C CH 2)nH

Most often n = 10 Free CoQ can undergo a 2 eoxidation/reduction: Q + 2 e- + 2 H+ QH2.

coenzyme QH2

Coenzyme Q (CoQ, Q or ubiquinone) is lipid-soluble. It dissolves in the hydrocarbon core of a membrane.

the only electron carrier not bound to a protein.

it can accept/donate 1 or 2 e-. Q can mediate etransfer between 2 e- that transfer and 1 ecarriers

Cytochromes
NAD+ FMN FeS FAD FeS ubiquinone Cyt b ubiquinone FeS Cyt c1 Cyt c Cyt a Cyt a3 1/2 O2

proteins that accept electrons from QH2 or FeS

Ultimately transfers the electrons to oxygen

Cytochromes
Cytochromes are electron carriers containing hemes . Hemes in the 3 classes of cytochrome (a, b, c) differ in substituents on the porphyrin ring.

Some cytochromes(b,c1,a,a3) are part of large integral membrane protein complexes. Cytochrome c is a small, water-soluble protein.

Heme is a prosthetic group of cytochromes. Heme contains an iron atom in a porphyrin ring system.

The heme iron can undergo 1 e- transition between ferric and ferrous states: Fe3+ + e Fe2+ Copper ions besides two heme A groups (a and a3) act as electron carriers in Cyta,a3 Cu2++e- Cu+

Electron carriers
NAD+, flavins and Q carry electrons and H+ Cytochromes and non-haem iron proteins carry only electrons

NAD+ FAD undergoes only a 2 e- reaction; cytochromes undergo only 1e- reactions FMN Q undergoes 1e- and 2 e- reaction

Electron Transport chain (respiratory chain)

The electron transport chain in the inner mitochondrial membrane can be isolated in four proteins complexes(I, II, III, IV). A lipid soluble coenzyme (Q) and a water soluble protein (cyt c) shuttle between protein complexes Electrons transfer through the chain - from complexes I and II to complex IV

Mitochondrial Complexes
NAD+ FMN

I
FeS FAD FeS ubiquinone Cyt b

NADH Dehydrogenase

II
Succinate dehydrogenase
ubiquinone

Cytochrome Oxidase
FeS Cyt c1 Cyt c Cyt a Cyt a3 1/2 O2

III
CoQ-cyt c Reductase

IV

The electron transport chain Mitochondrial Complexes

NADH:Ubiquinone Oxidoreductase Complex I

One of the largest macro-molecular assemblies in the mammalian cell Over 40 different polypeptide chains, encoded by both nuclear and mitochondrial genes NADH binding site in the matrix side Non-covalently bound flavin mononucleotide (FMN) accepts two electrons from NADH Several iron-sulfur centers pass one electron at the time toward the ubiquinone binding site

NADH:Ubiquinone Oxidoreducase is a Proton Pump

Transfer of two electrons from NADH to uniquinone is accompanied by a transfer of protons from the matrix (N) to the inter-membrane space (P)
Experiments suggest that about four protons are transported per one NADH NADH + Q + 5H+N = NAD+ + QH2 + 4 H+P

Reduced coenzyme Q picks up two protons

Succinate Dehydrogenase Complex II

FAD accepts two electrons from succinate Electrons are passed, one at a time, via ironsulfur centers to ubiquinone that becomes reduced QH2

Complex II: succinate-coenzyme Q reductase (succinate dehydrogenase)

The only TCA cycle enzyme that is an integral membrane protein, also called flavoprotein (FP2). Succinate fumarate + 2H+ + 2eUQ + 2H + + 2e- UQH2 Components: FAD and Fe-S centers No proton pumped

Cytochrome bc1 Complex Complex III

Uses two electrons from QH2 to reduce two molecules of cytochrome c CoQ passes electrons to cyt c (and pumps H+) in a unique redox cycle known as the Q cycle

The Q Cycle

Experimentally, four protons are transported across the membrane per two electrons that reach cyt. c Two of the four protons come from QH2 The Q cycle provides a good model that explains how two additional protons are picked up from the matrix

Cytochrome Oxidase Complex IV

Mammalian cytochrome oxidase is a membrane protein with 13 subunits Contains two heme groups Contains copper ions

Two ions (CuA) form a binuclear center Another ion (CuB) bonded to heme forms FeCu center

48

49

Order and Reduction Potentials

NAD+ FMN FeS FAD FeS ubiquinone Cyt b ubiquinone FeS Cyt c1 Cyt c

-0.32

-0.3

+0.045 +0.077

+0.03

+0. 29
Cyt a

+0. 55
Cyt a3 1/2 O2

+0. 22

+0. 25

+0.82

Peter D. Mitchell proposed the chemiosmotic hypothesis in 1961.The theory suggests essentially that most ATP synthesis in respiring cells come from the electrochemical gradient across the inner membranes of mitochondria by using the energy of NADH and FADH2 formed from the breaking down of energy rich molecules such as glucose.

Molecules such as glucose are metabolized to produce acetyl CoA as an energy-rich intermediate. The oxidation of acetyl CoA in the mitochondrial matrix is coupled to the reduction of a carrier molecule such as NAD and FAD. The carriers pass electrons to the electron transport chain (ETC) in the inner mitochondrial membrane, which in turn pass them to other proteins in the ETC. The energy available in the electrons is used to pump protons from the matrix across the inner mitochondrial membrane, storing energy in the form of a transmembrane electrochemical gradient. The protons move back across the inner membrane through the enzyme ATP synthase. The flow of protons back into the matrix of the mitochondrion via ATP synthase provides enough energy for ADP to combine with inorganic phosphate to form ATP. The electrons and protons at the last pump in the ETC are taken up by oxygen to form water.

This was a radical proposal at the time, and was not well accepted. The prevailing view was that the energy of electron transfer was stored as a stable high potential intermediate, a chemically more conservative concept.

The problem with the older paradigm is that no high energy intermediate was ever found, and the evidence for proton pumping by the complexes of the electron transfer chain grew too great to be ignored. Eventually the weight of evidence began to favor the chemiosmotic hypothesis, and in 1978, Peter Mitchell was awarded the Nobel Prize in Chemistry.
Chemiosmotic coupling is important for ATP production in chloroplasts and many bacteria.

The proton-motive force

In all cells, chemiosmosis involves the proton-motive force (PMF) in some step. This can be described as the storing of energy as a combination of a proton and voltage gradient across a membrane. The chemical potential energy refers to the difference in concentration of the protons and the electrical potential energy as a consequence of the charge separation (when the protons move without a counter-ion).In most cases the proton motive force is generated by an electron transport chain which acts as both an electron and proton pump, pumping electrons in opposite directions, creating a separation of charge. In the mitochondria, free energy released from the electron transport chain is used to move protons from the mitochondrial matrix to the intermembrane space of the mitochondrion.

Moving the protons to the outer parts of the mitochondrion creates a higher concentration of positively charged particles, resulting in a slightly positive, and slightly negative side (then electrical potential gradient is about -200 mV (inside negative). This charge difference results in an electrochemical gradient. This gradient is composed of both the pH gradient and the electrical gradient. The pH gradient is a result of the H+ ion concentration difference. Together the electrochemical gradient of protons is both a concentration and charge difference and is often called the proton motive force (PMF). In mitochondria the PMF is almost entirely made up of the electrical component but in chloroplasts the PMF is made up mostly of the pH gradient. In either case the PMF needs to be about 50 kJ/mol for the ATP synthase to be able to make ATP

ATP synthase
Proton diffusion through the protein drives ATP synthesis!
Two parts: F1 and F0 (latter was originally "F-o" for its inhibition by oligomycin) ---F1: ATP synthesis, a3b3gde ---Fo: Proton channel in inner membrane, ab2c10-12 Binding change mechanism: the three catalytic b subunits are intrinsically identical but are not functionally equivalently at any particular moment where they cycle through three conformational states.

Mitochondrial ATP Synthase Complex

The proton-motive force causes rotation of the central shaft g
This causes a conformational change within all the three ab pairs The conformational change in one of the three pairs promotes condensation of ADP and Pi into ATP

NARP syndrome : point mutation at nucleotide 8993 on the gene encoding subunit 6 of mitochondrial ATP Synthase.

The symptoms included developmental delay, retinitis pigmentosa, dementia, seizures, ataxia, proximal neurogenic muscle weakness, and senory neuropathy, in a pedigree consistent with maternal transmission .
INHIBITORS AND UNCOUPLERS

INHIBITORS OF ETC
COMPLEX I: COMPLEX IV COMPLEX II COMPLEX III

Rotenone Cyanide,H2S
Amobarbital

Carboxin
TTFA

Antimycin

Inhibitors of electron transport and oxidative phosphorylation

Complex IV

Complex III Complex I or III

Drugs that inhibit the ETC

NAD+

Amytal I rotenone
FAD FeS

FMN FeS ubiquinone Cyt b

Rotenone helps natives of the Amazon rain forest catch fish!

II
Antimycin A
ubiquinone

CN- CO
binding tightly to the ferric form (Fe3+) of a3

FeS

Cyt c1

Cyt c

Cyt a

Cyt a3 1/2 O2

III

IV

Inhibitors and Artificial Electron Acceptors

Rotenone amytal

Antimycin A

CN-,CO

Methylene blue +0.01 Ferricyanide+0.36

INHIBITORS OF OXIDATIVE PHOSPHORYLATION ATRACTYLOSIDE(BLOCK MOVEMENT OF ATP & ADP) OLIGOMYCIN(BLOCK FLOW OF H+ THROUGH Fo)

BONGREGATE(SAME AS ATRACTYLOSIDE)
IONOPHORE (LYPOPHILIC MOLECULE) example: 1. VALINOMYCIN (MOBILE ION CARRIER), 2. GRAMICIDIN (CHANNEL

FORMER)

UNCOUPLERS OF OXIDATIVE PHOPHORYLATION

2,4 DINITROPHENOL 2,4 DINITROCRESOL CCCP (MOST ACTIVE)chloro carbonyl cyanide phenyl hydrazone DICOUMAROL(vit. K analogue) VALINO MYCIN CALCIUM

PHYSIOLOGICAL UNCOUPLERS

UCP1(THERMOGENIN) ACTIVATION OF FATTY ACID OXIDATION HEAT PRODUCTIO IN BROWN ADIPOSE TISSUE EXCESSIVE THYROID HORMONE EFA DEFICIENCY LONG CHAIN FATTY ACIDS IN ADPOSE TISSUE CONJUGATED HYPERBILIRUBINEMIA

Reduction Potentials
E0=standard reduction potential. The relative tendency to accept e-s and become Crucial equation: reduced.

Go' = -nF Eo'

Number of electrons = Eo'(acceptor) - Eo'(donor) transferred in the Faradays constant redox reaction (96485 J/volt/mole)

If Eo' is positive, an electron transfer reaction is spontaneous (Go' <0)

Fumarate+2H++2e- succinate FAD+ 2H++2e- FADH2 NAD++ 2H++2e- NADH+H+

0.031 0 -0.32

Succinate+FAD Fumarate+ FADH2 G' = - n F E' Eo' = Eo'(acceptor) - Eo'(donor) G =-2*96485*(0-0.031)= 5.98KJ/mol Succinate+ NAD+ Fumarate+ NADH+H+ G =-2*96485*(-0.32- 0.031)= 67.7KJ/mol

Removal of H across a C-C bond is not sufficiently exergonic to reduce NAD+,but it does yield enough energy to reduce FAD. Thats why succinate dehydrogenase uses FAD other than NAD+ as coenzyme.