8 april 2008 electron transport, concluded; light reactions of photosynthesis andy howard...

8 April 2008

Electron Transport, concluded;

Light reactions of Photosynthesis

Andy HowardIntroductory Biochemistry

8 April 2008

8 April 2008 ETS and Photosynthesis p. 2 of 63

Proton translocation

Phosphorylation of ADP in mitochondria and in chloroplasts gets energy from translocation of protons across a gradient


What we’ll discuss Oxidative

Phosphorylation, concluded Complexes I-IV Complex V: Structure Proton leaks ATP translocation NADH translocation

Photosynthesis Light reactions: PS II Light reactions: PS I Output


Complexes I-IV There are several multi-enzyme complexes

involved in converting the reductive energy in NADH and FADH2 to their final products.# NameI NADH-Ubiquinone oxidoreductaseII Succinate-ubiquinone oxidoreductaseIII Ubiquinol-cytochrome c oxidoreductaseIV Cytochrome c oxidase


Overview of Oxidative Steps

Chart courtesyMichael King,Indiana State


Complex I

NADH:Ubiquinone oxidoreductase Embedded in inner mitochondrial

membrane Passes electrons from NADH to

ubiquinone 41 subunits (7 from mitochondrial genes)


Geometry & chemistry of complex I (fig. 14.7)

Roughly L-shaped multi-protein complex Segment inside mitochondrion accepts NADH’s

electrons FMN accepts those and passes them to an iron-

sulfur complex Iron-sulfur complex passes electrons to an

ubiquinone molecule in the inner mitochondrial membrane

Proton translocation occurs in connection with last step


Flavin cofactors

Participants in ETS

Sometimes depicted as starting points, but it’s probably better to think of them as intermediaries


Protons in Complex I

Complex I picks up a pair of protons from the matrix and passes them up through to the other side of the membrane

Energy for this translocation (against the concentration gradient) supplied by oxidation of NADH Diagram courtesy

of Rice University


Complex II Succinate-ubiquinone

oxidoreductase We’ve looked at this already as

succinate dehydrogenase Succinate + Q Fumarate + QH2

No protons translocated in this step:Reaction is close to isoergic

FAD and Fe-S proteins involved

PDB 2FBW246 kDadimer of hetero-tetamersChicken


Complex III Ubiquinol:cytochrome c oxidoreductase Transfers electrons from reduced ubiquinol

to iron atoms in cytochrome c One electron per cytochrome c Enzyme contains three main subunits:

cytochrome c1, cytochrome b,and an iron-sulfur (“Rieske”) protein

Net reactions: QH2 + cyto c–Fe3+ QH• + cyto c–Fe2++ H+

QH• + cyto c–Fe3+ Q + cyto c–Fe2+ + H+


Complex III structures

Cytochrome bc1 complex Contains two FeS

complexes & heme (c1) 11 polypeptides

Rieske Fe-S protein 3-layer beta sandwich With Fe2S2 cluster

PDB 2IBZ242 kDa hetero-undecameryeast

PDB 1RIE14 kDa monomerbovine


Cytochrome-dependent steps Cytochromes are, in general, proteins

involved in electron transport. Cytochrome c is a mobile, soluble

compound Others are generally membrane-

associated What they have in common:

covalently bound heme


Cytochromes The name derives from the fact that many of

them are colored and found in cells. In particular, cytochrome c, which is a

significant intermediary in the ETS, is a water-soluble, relatively small, heme-containing protein

Cytochrome c received substantial attention in the early years of biochemistry both because of its inherent importance and because it's easy to study.


Cytochrome c and evolution Cytochrome c is highly conserved;

the rate of mutation across the billions of years of evolution is remarkably slow, as compared to other proteins. This is generally a sign that its function is sufficiently irreplacable that even a modest modification in the protein renders the cell unviable.


Composite evolutionary tree

Schwartz & Dayhoff (1978) Science 199:395

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.


Cytochrome c structure Only 12kDa Heme group appears

between helices Covalent linkage to

heme visible here Mainly alpha helical

“orthogonal bundle”PDB 1AKK11.4 kDa monomerhorse heart


Complex 3: schematic

CourtesyU.Texas


Complex IV (fig. 14.12) Cytochrome c oxidase Transfers electrons from (soluble)

cytochrome c to molecular oxygen:Product is water

2Cyto c–Fe2+ + (1/2)O2 + 2H+ 2Cyto c–Fe3+ + H2O

Proton translocation: 4 protons emitted to outside per 4 molecules of cytochrome c (see eqn. 14.13); that’s 2 protons emitted outside per 2 molecules of cytochrome c

Therefore 2 protons translocated per 1 QH2 submitted to complexes III & IV


Cytochrome oxidase

2 functional units; Up to 13 subunits containing

membrane-spanning helices 4 protons produced per

oxidation of two molecules of QH2 at the Q0 site

See fig. 14.13

PDB 2EIJ367 kDa dimer ofheteroundecamersbovine


Cytochrome oxidase mechanism

Depends on two Cu+ ions

www.steve.gb.com


Mitochondrial sequestration Mitochondrion is a fairly complex organelle,

found in all eukaryotes. Some simple algae have one mitochondrion

per cell, whereas some protozoa have a half-million per cell.

A mammalian liver cell contains about 5000 mitochondria. These organelles resemble bacteria in size and complexity.

Substantial evidence exists to suggest that mitochondria are “captured” bacteria that have lost a lot of their initial genetic independence


Mitochondrial genetics Vertebrate mitochondrion has its own

chromosome, but it does not code for many proteins

Human mitochondrion codes for 17 proteins, plus two dozen specialized tRNAs and (presumably) some control elements.

Most of the ~1000 proteins that function in the mitochondrion are coded for in the host genome and are translocated, sometimes with some amount of proteolytic processing, from the ribosomal protein-synthesis mechanisms of the cytosol into the interior of the mitochondrion.


Mitochondrial structure

The mitochondrion has asmooth outer membraneand a highly convoluted innermembrane

Intermembrane space between them Outer membrane is permeable to small

molecules, so functionally the intermembrane space is equivalent to the cytosol.


Mitochondrial localization In eukaryotes, ETS and Krebs reactions

take place in the mitochondrion. Many reactions occur on the inner

membrane in its folded surfaces called cristae.

Localization to the membrane provides for orderly passage of substrates or electrons from one protein to the next, helping to defeat old man entropy.


Mitochondrial matrix enzymes

Some proteins do function in the matrix, the aqueous compartment of the mitochondrion interior to the inner membrane.

Pyruvate dehydrogenase complex TCA-cycle enzymes (except succinate

dehydrogenase, which is embedded in the inner membrane)

Some enzymes involved in fatty acid oxidation.


What’s the matrix like? This matrix is has such a high

overall protein concentration that it is not really an aqueous medium; it's a gel.

Think of reactions that occur in the mitochondrial matrix as occuring in Karo syrup (except the syrup is made of protein + H2O, not sugar + H2O)


Regulation of ETS An element of regulation of electron

transport is based on the availability of adenosine diphosphate, the substrate for the oxygen-mediated respiration steps. Mitochondria consume oxygen rapidly when ADP is available.


Uncouplers• When all the ADP in a

mitochondrion is consumed, rapid resiration stops.

• There are compounds that uncouple oxidation from phosphorylation

• when these uncouplers are added to a test system, it will continue to oxidize NADH even in the absence of ADP.

• 2,4-dinitrophenol is an example of an uncoupler.


ATP synthase Crucial example of a molecular motor,

i.e.,a machine that translates between chemical energy and mechanical work

Ultimately its job is to pull protons across the membrane, using the energy associated with that favorable translocation to drive the synthesis of ATP from ADP and Pi

The motor is what rotates the protein through various positions to enable its reactions

Image courtesyThomas Meier,MPI Frankfurt


Chemiosmotic theory: history How are these reactions actually

employed to drive ATP synthesis? This was hotly debated for many years. Problem was finally solved in 1960's:

chemiosmotic theory, which links ATP synthesis to proton translocation across the membrane

Oxidations in complexes I, III, and IV is what drove the protons against a concentration gradient in the first place


The developers

Peter Mitchell, Paul Boyer,Ephraim Racker, and others.

Mitchell received the Chemistry Nobel Prize in 1978; Boyer received his nineteen years later, to the immense satisfaction of those who felt he had been passed over the first time around.


Chemiosmotic hypothesis Recognizes that protons have

been pushed across the membrane against the concentration gradient

Flow back across generates energy, which is harnessed to perform rotations of the ATP synthase molecule, driving synthesis of ATP


Structural realities F0 component is

transmembrane helical domain:Grabs protons,channels them inward

F1 is the motor Homotrimeric (3*(+)) At any moment, each

subunit is in one state, and then moves to the next state


ATP Synthase: Details ADP and Pi bound to one subunit

(the open site) Fully-formed but bound ATP attached at second

subunit (the loose site) ATP ready for release located in third subunit

(the tight site) After one event, the entire complex rotates 120º;

process continues

Diagram courtesy of Gonzaga U.


ATP Synthase mechanism

Careful explanation in section 14.9;attend to it!

130 revolutions per second observed in model studies with actin attached to the gamma (rotating) subunit


ATP Synthase Other molecular

motors exist; this is probably the best-characterized

PDB 2HID1136 kDa,trimer of (9-subunit complexes)yeast


Proton Leaks If NADH oxidation is uncoupled

physiologically from ATP synthesis, heat is produced

This happens on purpose in homeotherms, particularly infants and hibernating animals

Uncoupling caused by thermogenin (UCP1), a channel-making protein


Mechanism of thermogenin

External Signal (cold?) Norepinephrine binds to external receptor adenylyl cyclase activation (ATP -> cyclic AMP) activation of protein kinase A phosphorylation of triacylglycerol lipase release of fatty acids activation of thermogenin so it leaks protons


Moving ATP to the cytosol Most ATP used in cytoplasm for anabolic

reactions Need a transporter to get it out:

adenine nucleotide translocase ATP,ADP uncomplexed to Mg2+ here Net loss of charge of -1 in matrix results This powers the translocation but slightly

impoverishes the gradient

Need a phosphate transporter inward too:phosphate symport with H+


P/O Ratio Common descriptor for the number of ATP-

synthetic reactions that can be accomplished per atom of oxygen that gets converted to water.

P/O for NAD-dependent reactions is 2.5 10 protons translocated per NADH

(4 in complex I; 4 in complex III; 4/2=2 in complex IV) 1 ATP produced for 4 protons flowing back So 10 / 4 = 2.5 ATP per NAD reaction

P/O for Q-dependent reactions is 1.5:Only 6 protons in per QH2: 6/4=1.5.


NADH Shuttle mechanism I:glycerol phosphate shuttle

Fig.14.18

Cartoon courtesy Indiana State U.


NADH transport II:malate-aspartate shuttle

Fig. 14.19

Cartoon courtesy Leeds dentistry


Other terminal electron acceptors, donors Note that life precedes both the evolution

of photosynthesis and the appearance of high [O2] in the atmosphere

Chemiautotrophic bacteria depend on oxidizing H2, NH4

+, NO2-, H2S, S, or Fe2+

for energy production E.coli can use fumarate as terminal

acceptor


Oxidative damage Production of radicals is essentially

unavoidable when O2, H2O present and redox reactions occur

Particular problem is superoxide, O2-•

Obligate anaerobes lack protection against these species, which arise whenever oxygen is involved in redox reactions in aqueous media

Aerobic organisms find ways to detoxify superoxide and related species


Superoxidedismutase

Variety of structural forms; all involve metal ions

Reaction is2O2

-• + 2H+ H2O2 + O2

PDB 1DSWCu-Zn17kDamonomerhuman

PDB 1PL4MnSOD87kDa tetramerhuman


What happens to the resulting peroxide?

Peroxide itself is somewhat toxic

So it gets further degraded via catalase, a heme enzyme

2H2O2 2H2O + O2

Often performed in the peroxisome

1E93213 kDa tetramerProteus mirabilis


Photosynthesis

Definition:harvesting of light to generate energy

Happens in plants, photosynthetic bacteria Photosynthetic reactions offer source for

carbon fixation as well as energy in photoautotrophs

In higher plants these events happen in the thylakoid disks of chloroplasts


Light reactions

Electrons are promoted from the ground state to excited states upon absorption of a photon by a chromophore

Drawing courtesy JohnsonCounty Community College


Chlorophyll Heme-like chromophore

with Mg in the center Absorbs strongly in red

and blue; therefore it appears green

Minor variations in side groups

www.steve.gb.com/science/photosynthesis.html


Chlorophyll absorption

Plot courtesy Davidson U. Biology Dept


Other light-absorbing pigments Chlorophyll in its various forms is not the

only light-absorbing pigment in plants and photosynthetic bacteria

Accessory pigments / accessory proteins involved in resonant energy transfers to chlorophyll

Examples: carotenoids (particularly -carotene), phycoerythrin, phycocyanin

-carotene; max = 497 nm


Accessory pigments in phycobilisomes

Water absorbs red light strongly Shorter wavelengths (higher energies) are more

penetrating Aquatic plants need accessory pigments that

absorb in the range that’s available Energy absorbed by phycobiliproteins is

transferred ultimately to ChlA by Förster resonances


Prosthetic groups in phycobiliproteins

Conjugations account for absorption maxima

Shown is phycocyanobilin, the prosthetic group from phycocyanin

PDB 1JBO240 kDa hexamer of heterodimers1 heterodimer shownSynechococcus elongatus


The special pair Two out of the large collection of chlorophyll

molecules within a single photosystem that are responsible for giving up electrons rather than just getting electrons excited into higher-energy states

Pair of P680 molecules in photosystem II are the special pair in that case(P870 in purple bacterium; P680 in cyanobacteria and green plants)

Other chlorophyll molecules and antenna molecules absorb photons too; these transfer energy to the special pair


Photosystem II Beginning of sequence of energy-generating

pathways in the chloroplast or the bacterial membrane

Involves P680, a chlorophyll positioned so that its absorption max is at 680nm

The version in purple bacteria is P870 Absorption maximum depends on

chromophore’s specific structure and on modulation by neighboring protein species


Chlorophyll a

P680 contains a pair of these chromophores, not covalently bound to proteins


Photosystem II structure Complex 18-mer Contains:

-carotene Chlorophyll A Heme Pheophytin A Quinones Lipids

PDB 2AXT602 kDa dimer of 18mersThermosynechococcus elongatus


Electron translocation in photosystem II

Two protons move across the thylakoid membrane for each electron promoted and transferred—plus two protons associated with the conversion of QH2 back to Q

This provide proton pumping capability like that in mitochondria

Difference: gradient is dependent only on pH difference, not electrical potential

Final electron acceptor is a ferredoxin-like Fe-S cluster


Photosystem I P700 is primary photon

acceptor Similar translocations of

protons Net reduction of NADP again Non-cyclic: we need to re-

oxidize Final electron acceptor is a

quinone

PDB 2O01347 kDa17 subunitsPisum sativumand other plants


Schematic of energy transfers in PS I

Drawing by J. Nield


Net light reactions

NADPH produced Eight protons pass across Energy is, in principle, available from

both sources, but NADPH is employed in anabolic reactions rather than as a source of ATP

Net ATP production per photon: unclear. Probably about 2.

8 april 2008 electron transport, concluded; light reactions of photosynthesis andy howard...

Documents

cytochrome cenzyme

cytochrome c1

proteincytochrome c

evolutioncytochrome

cytochrome cone electron

ubiquinone molecule

translocation of protons

complex icomplex