Module 0210101:
Molecular Biology and Biochemistry of the Cell
Lecture 17
Electron Transport and Carbon
Fixation in Chloroplasts
Dale Sanders
10 March 2009
Objectives
By the end of the lecture you should understand:
1.
3.
What are the identities of the components of the photosynthetic
electron transport chain.
How electron transport occurs between these components in terms
of
(a) their redox potentials (E’o);
(b) their distributions in the membrane;
(c) associated H+ flux.
The nature of cyclic electron transport.
4.
How CO2 is initially fixed by Rubisco.
5.
How 3-PGA is assimilated to hexose.
2.
Reading
As in my previous lectures, all the topics today are well
covered by the standard big biochemistry textbooks.
One such text is
Voet & Voet (2004) Biochemistry (3rd Ed.) Chapter
24 and especially pp. 884-895
Also useful for a more in-depth treatment is
Nicholls, DG & Ferguson, SJ (2002) Bioenergetics 3
Chapter 6.
Buchanan BB et al. (2000) Biochemistry and
Molecular Biology of Plants. Chapter 12,
pp. 568-590.
PS II and PS I act in Series to Catalyse
Electron Flow Between H2O and NADP+
Pathway of electrons from H2O
NADP+ :
NADP+ + H+
H2O
PS II
Redox
components
½O2
+2H+
PS I
Redox
components
NADPH
Light and Resonance
Energy Transfer
Light and Resonance
Energy Transfer
The Identities of the Redox Components of the
Thylakoid Electron Transport Chain:
Overview of redox chain components
-1.0
(FeS)
*P680
Pheophytin
E'o (V)
*P700
Fd
PQ
0
cytb
cytb
FeS
cytf
H2O
+1.0
Fp
NADP+
PC
P700
OEC P680
OEC = Oxygen–evolving complex; PQ = Plastoquinone
PC = Plastocyanin: Fd = Ferredoxin
Fp = Flavoprotein (FAD)
A more detailed look at the redox chain
components
1.
PS II Reaction Centre
Comprises a supramolecular complex:
several distinct proteins binding redox
chain components:
(i) P680: Chl a dimer
(ii) Pheophytin (Chl a without Mg2+) E'0  – 0.55 V
(iii) 2 molecules of plastoquinone, bound to specific
proteins. QA, QB
tight
loose
Plastoquinone (PQ)
(
oxidized (quinone)
(
reduced (quinol)
Oxidation of QB is prevented by a number of herbicides
(e.g. dichlorophenyldimethylurea (DCMU))
Once reduced to plastoquinol (PQH2) the QB molecule
diffuses into
2. The PQ Pool: A large number of molecules of PQ,
freely dissolved in the hydrophobic portion of the
thylakoid membrane.
3.
The Cytochrome b6f complex
A supramolecular complex, accepting e- from PQ
comprises:
-
2 spectroscopically–distinct b-type
cytochromes (cytochromes b6)
Cytochrome f
an Fe2S2 centre (Rieske protein)
bound PQ
Structurally and functionally, the cytochrome b6f
complex is very similar to Complex III (Cytochrome bc1)
of mitochondria:
(i)
(ii)
(iii)
(iv)
both are inhibited by Antimycin A
both accept e- from a quinol
Cyt f is similar structurally to cyt c1
the b-type cytochrome (actually 2 haem groups
bound to a single apoprotein) shows extensive
sequence homology to cyt b of mitochondria
(v) both contain a “high potential” (E’0 = + 300 mV)
Fe2S2 centre
All these factors point to a common evolutionary origin
of Complex III and the Cytochrome b6f complex
4. Plastocyanin (PC)
A small, water-soluble copper-containing
protein located in lumen of thylakoid.
5. PS I Reaction Centre – Oxidizes PC
The 3rd supramolecular complex,
comprising (bound to proteins):
(i)
(ii)
(iii)
(iv)
P700: Chl a dimer
6 additional Chls
2 quinones,
3 Fe4S4 centres
All help move
electrons across
membrane to next
component …
6. Ferredoxin (Fd)
A small protein with an Fe2S2 centre. Loosely
associated with the STROMAL side of the
thylakoid membrane.
7. Ferredoxin – NADP oxidoreductase
A flavoprotein, containing FAD
Also located on the STROMAL side of the
thylakoid membrane.
8. Oxygen-evolving complex – OEC
3 proteins; associated with PS II on
LUMINAL side of membrane
Active centre: 4 tightly-bound Mn2+ ions
Catalyses reaction:
2H2O
O2 + 4H+ + 4 e-
The e- are passed, one at a time via tyrosine
residues, to oxidized P680+ reaction centres.
The Useful Products of Photosynthetic e- Transport
1. NADPH:
2. PMF:
Subsequently used in reduction of CO2
e- transport chain pumps H+ into lumen, hence ATP
is synthesised
Magnitude of PMF
Δψ = +20 mV
ΔpH = 3.5 units (lumen acid)
Since PMF = Δψ + 59 (pHo – pHi)
Thus PMF = + 20 + 206 = + 226 mV (lumen + ve)
Note: PMF is inverted compared with mitochondria…
and so is orientation of ATP synthase: ATP made
on outside of thylakoid membrane, in stroma
STOICHIOMETRIES: For redox chain, 6H+/ 2eFor ATP synthase, 4H+/ATP
i.e. for each pair of e- passing through chain:
1 NADPH and 1.5 ATP are produced
Cyclic e- Transport and Variable
ATP/NADPH Production
Observation: Light of wavelength >680 nm results in a PMF,
but not net production of reducing equivalents.
Interpretation:
Stroma
Lumen
Interpretation: PSI is excited by long wavelength
light.
Electrons are recycled through ferredoxin, b6f complex
and plastocyanin b6f complex is a H+ pump
Note: Net production of NADPH is not possible because
no reductant (i.e. H2O ) is available.
But ATP can be produced.
Cyclic electron transport might provide plants with a way
of producing >1.5 ATP/NADPH if demand for ATP is
high.
Lateral Heterogeneity and Plastoquinone
Diffusion
Cyt b6f complex and PS I are in stromal lamellae;
PS II is in granal lamellae
Question: How are reducing equivalents
transferred from PS II to Cyt b6f complex??
Answer: Plastoquinone: A very mobile molecule,
which diffuses in the plane of the membrane.
PQH2
PQH2
PSI
PSII
Carbon Fixation
The 1st reaction: catalysed by Ribulose 1,5bisphosphate carboxylase/oxygenase (Rubisco)
–C–O–P
C=O
CO2
+
–C–O–P
–C–O–P
C – OH
C – OH
Rubisco
– C – OH
– C – OH
+
C
O
O–
C
O
O–
–C–O–P
Ribulose 1,5 bisphosphate
(3 – phosphoglycerate) x 2
Rubisco
Reaction Energetics: ΔGO = - 52 kJ/mol, hence spontaneous
The Protein: Very low turnover rate (about 3 s–1)
hence very abundant …
THE most abundant in the world
A large, allosteric enzyme:
8 large (L) subunits ….. Mr = 55,000
8 small (S) subunits …..Mr = 13,000
L subunits
S subunits
http://4e.plantphys.net/article.php?ch=6&id=78
Control: Mg2+ released from
thylakoid lumen in exchange for H+
during electron transport activates
Rubisco in stroma
Assimilation of Hexoses
3 – Phosphoglycerate
ATP
Phosphoglycerate kinase
ADP
1,3 – Bisphosphoglycerate
NADPH
Glyceraldehyde 3–Phosphate DH … NADP+ - specific
NAD+
Glyceraldehyde 3 –Phosphate
Dihydroxyacetone phosphate
Aldolase
Fructose 1,6-Bisphosphate
Pi
Fructose 1,6-Bisphosphatase
Fructose 6-Phosphate
Note:
1. These stromal reactions are a reversal of
glycolysis, except that FBPase provides a
unique step.
2. These reactions mirror those of
gluconeogenesis (in liver) except that
Glyceraldehyde 3-phosphate
dehydrogenase is NADP+-specific.
SUMMARY
1. The photosynthetic e- transport chain comprises
3 macromolecular complexes (PS II, Cyt b6f
complex, PS I) and associated redox
components.
2. PHS e- transport chain components can be
arranged according to E’O and to position in
membrane.
3. PHS e- transport chain is a H+ pump.
5. Cyclic e- transport involves PS I and cyt b6f:
ATP production but no reductant.
6. Rubisco catalyses CO2 fixation leading to
formation of PGA.
7. PGA is assimilated to hexose in stromal
reactions analogous to gluconeogenesis.