Mitochondria & Chloroplasts II

 Lecture Outline:

I.    Strategy of Photosynthesis (PS) in Plants

    A.    Photoreduction: light energy is trapped by chlorophyll and used to remove electrons
           and protons from water, forming O₂.

• The electron transport chain (ETC) is in the thylakoid membrane of chloroplasts, and the ETC includes two functionally and spatially distinct photosystems (PS).  Each photosystem is independently activated by light, but electrons flow from PS II to PS I in a process of linear electron flow, during which electrons are transferred from water to NADP⁺.
• NADPH is formed on the stromal side of the thylakoid membrane; NADPH then diffuses into the stroma to be used in Calvin-Benson cycle (see C below).
• Electron movement through the ETC provides energy to actively transport protons from stroma into thylakoid lumen.  Resulting proton gradient used to synthesize ATP (see B below).

    B.    Photophosphorylation:

• ATP synthesis due to protons moving down their concentration gradient from the intrathylakoid space into the stroma through the chloroplast ATP synthase(CF₀CF₁complex).
• ATP is synthesized on the stromal surface of the thylakoid membrane. It then diffuses into the stroma to provide energy to drive the Calvin-Benson cycle (see C below).

    C.    Carbon-fixation:  ATP and NADPH are used to fix CO₂

• CO₂ added to ribulose 1,5-bisphosphate (5C); ATP and NADPH are then used to produce two molecules of glyceraldehyde-3-phosphate (3C),which can be transported into the cytosol for the synthesis of sucrose and other molecules.  The subsequent reactions of the Calvin cycle use the remainder of the glyceraldehyde-3-phosphate to regenerate the ribulose1,5-bisphosphate.
• Known as Calvin-Benson cycle (a.k.a. dark cycle, since only steps 1 and2 require light) and occurs in stroma.

Note that Steps A and B require light, therefore also known as light reactions or photosynthetic electron transfer reactions.

II.    Similarities and Differences between Chloroplasts and Mitochondria

    A.    Similarities between Mitochondria (M) and Chloroplasts (C)

• Both have double membranes: porins in outer membranes allow ~free diffusion of molecules <5000 MW; inner membranes are permeability barrier and contain transport proteins that enable only certain substances to cross.
• ATP generated by chemiosmotic coupling in both.  Process includes:

- redox reactions/ sequential movement of electrons along electron-transport chain (ETC)
- energy of favorable electron transport used to transport protons across membrane
- H⁺ gradient used by ATP synthase to synthesize ATP

• Both have physical/functional separation between:

(1) chemiosmotic processes that occur on membranes and utilize NADH/O₂(M) or H₂O (C) and produce H₂O (M) or NADPH/O₂(C)   versus
(2) metabolic pathways (citric acid cycle or Calvin cycle) that occur in the matrix (M) or stroma (C) and use sugars and produce CO₂(M) or use CO₂ and produce carbohydrate (C).

    B.    Differences between Mitochondria (M) and Chloroplasts (C)

• Net result of photosynthetic electron transfers in chloroplasts is to drive electrons "uphill":  photon capture by PSII and PSI is used to energize electrons such that their net flow is from H₂O(low reduction potential/"poor" electron donor) to NADP⁺(high reduction potential/"poor" electron acceptor).
• Products of electron transport and chemiosmotic coupling (ATP and NADPH) are consumed in "dark" reactions in stroma of chloroplast, which are catalyzed by enzymes of Calvin cycle.  Citric acid cycle in mitochondria provides NADH to the ETC, and the ATP produced by chemiosmotic coupling is transported out of the mitochondrion for use in the cytosol.
• Chloroplasts have an extra compartment:  thylakoid membrane (not ICM) is site of ETC and ATP synthase.
• ETC of chloroplast has four redox complexes (PS II, PS I cytochrome b/f [which is a H⁺ pump] and FAD/NADP⁺ reductase) and three mobile electron carriers (plastoquinone, plastocyanin, and ferredoxin).  The FAD/NADP⁺ reductase is a peripheral membrane protein complex on the stromal surface of the thylakoid membrane.
• Protons are pumped out of stroma into thylakoid space in chloroplasts.
• Since the thylakoid membrane is permeable to ions such as Mg²⁺and Cl^-, flow of these ions dissipates most of the membrane potential.  Thus, almost all of the proton motive force in chloroplasts is due to the pH gradient across the thylakoid membrane (~pH 8.0 in stroma and ~pH 5.0 in thylakoid lumen).  Mitochondria presumable need a large membrane potential because they could not tolerate having their matrix at pH 10, as would be required to generate their proton-motive force without one.

Those compartments with a similar pH have been colored similarly in the above figure.  Note that ATP synthase is similarly arranged inboth organelles, such that the F₁ region is in a pH 8 environment in both (the matrix or stroma, respectively).  Thus, the catalytic site for ATP synthesis is at a similar pH in both organelles.
III.    Molecular Analysis of Photosynthesis 

    A.    Structure and Function of Chlorophylls

• Chlorophyll molecules consist of a porphyrin ring attached to a long hydrocarbon side chain; porphyrin ring contains a central Mg²⁺ ion (rather than Fe atom of heme) and an additional five-membered ring fused to pyrrole ring III; different chlorophylls vary in structure of certain side groups of porphyrin ring.
• When a chlorophyll molecule absorbs a photon of light, one of its electrons is promoted from its ground (lowest energy) state molecular orbital to one of higher energy, thereby resulting in an "excited" chlorophyll molecule.
• An electronically-excited chlorophyll molecule is unstable, and it will dissipate its excitation energy (thereby returning to its unexcited ground state) in a variety of ways.
• Decay by giving off light (fluorescence) and heat (1 in figure below).
• Decay by resonance energy transfer, in which an excited molecule directly transfers its excitation energy to nearby unexcited molecules with similar electronic properties (2 in figure below).
• Photooxidation, in which a light-excited (donor) molecule is oxidized by transferring an electron to an acceptor molecule, which is thereby reduced.  Electronic energy is converted to chemical energy in this process (3 in figure below).

• Chlorophyll molecules are arranged in multi-protein photosystems in the thylakoid membrane such that the second and third processes happen most of the time.  In plant cells, only the two molecules of chlorophyll a that are in the reaction centers of the photosystems can carry out photooxidation.  The surrounding antenna complex (containing one or more light-harvesting complexes) contains chlorophylls a and b and other pigments, which funnel light energy to the reaction centers through resonance energy transfers.

• Model of photochemical charge separation in the reaction centers: excitation of chlorophyll a at the luminal side of the reaction center is followed by the transport of an electron from the excited chlorophyll to a primary electron acceptor that is bound to the stromal surface of the reaction center.  Following electron transfer, the primary electron acceptor is negatively charged and becomes a strong reducing agent and the chlorophyll a molecule is positively charged and becomes a strong oxidizing agent.  The transferred electron must then be replaced in the oxidized chlorophyll to return it to its ground state.

B.    Chloroplasts contain two functionally and spatially distinct photosystems (PS).  Each photosystem is independently activated by light, but electrons flow from PS II to PS I in a process of linear electron flow, during which electrons are transferred from water to NADP⁺.

• PS II (or P680) is excited by slightly shorter wavelength of light; the primary electron acceptor of PS II is plastoquinone (Q), and water is the source of electrons that replace the transferred electron of the reaction center chlorophylls in PS II.
• Electrons are stripped one at a time from two molecules of H₂Oby the oxygen-evolving complex, which is on the luminal surface of PS II's reaction center.  This complex is unusual in that manganese ions are used to carry out this process.
• The protons that are left after water is stripped of its electrons remain in the thylakoid lumen, contributing to the proton gradient across thethylakoid membrane.
• Reduced plastoquinone (QH₂) diffuses back across the thylakoid membrane to the luminal surface and donates its two electrons to the cytochrome b/f complex.  In the process, QH₂ shuttles two protons across the membrane, adding to the proton gradient.
• Electron flow through the cytochrome b/f complex can also result in proton translocation across the thylakoid membrane, adding to the proton gradient.
• Plastocyanin is a soluble electron carrier on the luminal surface of the thylakoid, and it shuttles electrons from the cytochrome b/f complex to the reaction center of PS I.  PS I is driven by light of wavelength700 nm or less.  Its excitation results in the reduction of the diffusible electron carrier ferredoxin on the stromal surface of the thylakoid membrane. The electrons are transferred to FAD and then to NADP⁺ via the action of NADP⁺-FAD reductase, a multi-subunit redox complexon the stromal surface of the membrane.
• The PS I chlorophyll is restored to its ground state by the electron it receives from plastocyanin.

C.    Protons flow from the thylakoid lumen into the stroma, primarily driven by the large concentration difference (pH 8 in stroma vs. pH 5 in lumen).  Chlorophyll ATP synthase uses proton flow to synthesize ATP from ADP and P_i, similar to process in mitochondria.

D.    ATP and NADPH are both generated on the stromal side of the thylakoid membrane and are subsequently used in the Calvin-Benson cycle to fix CO₂ to make sugars.

• First reaction is the addition of CO₂ to the 5-C molecule ribulose1,5-bisphosphate.  This reaction is catalyzed by rubisco (ribulose1,5-bisphosphate carboxylase, and results in the formation of two molecules of the 3-phosphoglycerate (3-C).
• In the next two steps, ATP and NADPH are used to convert 3-phosphoglycerate to glyceraldehyde-3-phosphate, which can be bled out of the cycle and transported into the cytosol to make sucrose and other molecules.
• The rest of the Calvin cycle uses the remainder of the glyceraldehyde-3-phosphatemolecules to regenerate ribulose 1,5-bisphosphate.