Oxidative Phosphorylation and Photophosphorylation: Structure, Mechanism, and Regulation

Oxidative Phosphorylation

Introduction

Oxidative phosphorylation is the process by which cells generate ATP using energy derived from the transfer of electrons through a series of protein complexes in the mitochondria. This process is central to cellular metabolism and was explained by Kennedy and Lehninger in 1948. It involves redox reactions and the creation of chemical gradients, ultimately channeling free energy into the formation of ATP.

• Location: Takes place in the mitochondria.

• Goal: To make ATP, the universal energy currency of the cell.

• Mechanism: Uses redox reactions and chemical gradients.

Overview of the Process

• Electron Flow: Electrons flow through a series of membrane-bound proteins, originating from metabolic pathways such as the TCA cycle and glycolysis.

• Coupling: Exergonic electron flow is coupled to endergonic proton flow, conserving energy as a membrane potential.

• ATP Formation: Proton flow back across the membrane (exergonic) allows free energy to be used in ATP synthesis.

Mitochondrial Structure

The mitochondrion has an outer membrane, an inner membrane with cristae, and a matrix. The inner membrane is the site of the electron transport chain and ATP synthesis.

• Inner Membrane: Contains protein complexes for electron transport and ATP synthesis.

• Matrix: Site of the TCA cycle and contains enzymes for metabolism.

• Intermembrane Space (IMS): Accumulates protons during electron transport.

Oxidative Phosphorylation

• Generating energy through the flow of electrons

• These electrons are shuffled via electron acceptors

• NAD or NADP are reversibly bound to proteins and can accept 2 electrons via a hydride

• FAD or FMN are often tightly bound to the enzyme and can accept 1 or 2 electrons

Other electron carriers

• NAD/FAD are the major electron carriers in cells

• 3 other types of electron carriers are associated with the membrane-bound In mitochondrial OP pathway

– Ubiquinone (coenzymeQ)

– Cytochromes

– Iron-Sulfur Proteins

Electron Transport Chain (ETC)

Main Complexes and Electron Carriers

The ETC consists of four main complexes and several mobile electron carriers:

• Complex I (NADH Dehydrogenase): Transmembrane oligomer with 42 subunits, 1 FMN, and ~6 iron-sulfur clusters. Catalyzes transfer of electrons from NADH to ubiquinone (Q), pumping 4 protons into the IMS.

• Complex II (Succinate Dehydrogenase): Complex of 4 proteins, contains 3 Fe-S clusters, FAD, and a heme group. Transfers electrons from succinate to ubiquinone but does not pump protons.

• Complex III (Cytochrome bc1 Complex): Catalyzes transfer of electrons from ubiquinol (QH2) to cytochrome c, pumping 4 protons into the IMS.

• Complex IV (Cytochrome c Oxidase): Contains 3 hemes and 2 Cu atoms. Transfers electrons from cytochrome c to O2, producing water and pumping 2 protons into the IMS.

Electron Carriers

• NAD+/NADP+: Reversibly bound to proteins, accept 2 electrons via a hydride.

• FAD/FMN: Tightly bound to enzymes, accept 1 or 2 electrons.

• Ubiquinone (Coenzyme Q): Membrane-soluble, shuttles electrons between complexes. Exists in three forms: fully oxidized (Q), semiquinone radical (Q•), and fully reduced (QH2).

  • Benzoquinone containing a long, isoprenoid tail

    • Q10 (10 isoprenoids) is the most common in humans

  • Can exist in 2 reduced forms

  • Membrane soluble, so CoQ can shuttle electrons between larger, less mobile membrane bound proteins

• Cytochromes: Proteins with heme groups, shuttle electrons via changes in iron oxidation state. Classes: A (600 nm), B (560 nm), C (550 nm).

• Iron-Sulfur Proteins: Clusters coordinated by cysteine, histidine, or inorganic sulfur.

  • Reduction potential of these clusters can range from + to – depending on what is coordinating the Iron

Electron Transport Summary

The overall reaction for electron transport is:

• $\text{NADH} + 11\text{H}^+_{\text{matrix}} + \frac{1}{2}\text{O}_2 \rightarrow \text{NAD}^+ + 10\text{H}^+_{\text{IMS}} + \text{H}_2\text{O}$

Complex 1

• NADH Dehydrogenase

• Transmembrane oligomer of 42 subunits that acts as a proton pump

• 1 FMN and ~6 Iron-Sulfur clusters

• Catalyzes a 2-step reaction

  • Transfers a hydride from NADH and a proton from the matrix to Q

  • Transfers 4 protons from the matrix to the IMS

Complex 2

• Succinate Dehydrogenase - complex of 4 proteins (same protein involved in TCA)

• Transmembrane complex located in the inner leaflet of the inner membrane

• Contains 3 Fe-S clusters, an FAD, and a heme group

• Catalyzes the transfer of electrons from succinate to ubiquinone

Complex 3

• Cytochrome bc1 complex

  • 11 subunits not including Cytochrome C

  • Cytochrome C ≠ Cytochrome C1

• Catalyzes transfer of electrons from ubiquinol (QH2) to Cytochrome C

• Q is oxidized while cytochrome C1 is reduced.

• Each molecule of QH2 releases two protons into the IMS and donates its 2 electrons to cytochrome C1 and b, respectively

• Cytochrome C has a heme that accepts electrons from complex 3 and transfers it to complex 4

Complex 4

• Cytochrome C oxidase

• Contains 3 hemes and 2 Cu atoms. One in a Cu-Cys complex and one in a Cu-Heme complex

• Catalyzes the transfer of electrons from CytC to O2 and consumes H+ from the matrix

• Results in the production of water and release of protons into the IMS

Complex	Protons Pumped into IMS
Complex I	4
Complex II	0
Complex III	4
Complex IV	2

Proton Motive Force (PMF) and ATP Synthesis

Generation of PMF

The electron transport chain creates potential energy in two forms:

• Electrical Potential Energy: Separation of charge by driving H+ across the bilayer without counterions.

  • driving [H+] across the bilayer without counterions

• Chemical Potential Energy: Imbalance in H+ concentration across the membrane.

  • creating an imbalance in [H+]

The free energy change for proton movement is given by:

• $\Delta G = 2.3RT\Delta pH + ZF\Delta \psi$

ATP Synthase and Chemiosmotic Model

ATP synthesis in the mitochondria utilizes the pH and electrical gradients formed by the ETC as a source of energy. Protons re-enter the matrix by a proton-eclective pore, through ATP synthase, a complex protein with two major subunits (F0 and F1).

• F0 is a transmembrane homooligomer

consisting of • F1 is the soluble portion

• F0: Transmembrane homooligomer, forms a proton channel.

  • consists of proteins b2, a, , and 10 copies of C

• F1: Soluble portion, catalyzes ATP synthesis from ADP and Pi.

  • consists of 3a, 3b, 1g, and 1e proteins

This process is known as the chemiosmotic model.

Mechanism of ATP Synthase (F0F1 Catalysis)

• Rotational Catalysis: Protons from IMS protonate Asp in the C-ring, causing rotation and allowing a proton back into the matrix.

• Gamma Subunit: Rotation of the C-ring turns the gamma subunit, which interacts with the α/β complex.

• Conformational Changes: The three α/β dimers exist in three conformations: empty, ADP-bound, and ATP-bound. ATP is released only upon interaction of α/β with the gamma subunit.

  • Each turn forces gamma to interact with a different α/β dimer

Proton Motive Force Drives Other Reactions

Transport of ADP/ATP and Phosphate

• ADP/ATP Translocase: ADP (−3 charge) is pumped into the matrix, ATP (−4 charge) is pumped out to the IMS.

• Phosphate Translocase: Imports one H+ and one H2PO4− into the matrix for ATP synthesis.

  • The phosphate H2PO4- is used in the conversion of ADP to ATP

• The more negative species, ATP, is moving toward the more positive side of the membrane, the IMS

Regulation of Oxidative Phosphorylation

Control Mechanisms

• Energy production must be tightly regulated

• Acceptor Control: Regulation by levels of free ADP and Pi at the final step.

• Mass Action: Controlled by the equilibrium of products (ATP) and reactants (ADP and Pi).

Photophosphorylation (Photosynthesis)

Overview

Photosynthesis uses light energy to produce ATP in plants, algae, and some bacteria. It also involves electron transport reactions and consists of two divisions: light-dependent reactions and carbon fixation reactions.

• Location: Takes place in chloroplasts, which have thylakoid membranes containing photosynthetic pigments and ATP synthesis machinery.

• Reduction: Catalyzes the reduction of NADP+ to NADPH.

Chloroplast Structure

• Thylakoids: Membrane-bound compartments where light reactions occur.

• Stroma: Site of carbon fixation reactions.

Light Absorption and Photosystems

• Chlorophylls: Planar, polycyclic green pigments organized by proteins in the Light Harvesting Complex (LHC).

• Photosystems: Arrangements of chlorophylls and accessory pigments that funnel energy to photochemical reaction centers (PRCs).

Photochemical Reaction Centers

• Bacterial PRCs: Use pheophytin-quinone or quinone-Fe-S electron shuttles, consist of a reaction center, cytochromes, and ATP synthase.

• Plant PRCs: Two distinct photosystems (PSI and PSII). PSI uses iron-sulfur proteins; PSII uses a pheophytin-quinone system. Both converge at the cytochrome b6 complex.

Bacteriorhodopsin

• Structure: 7 transmembrane helices.

• Function: Light-driven proton pump, establishes H+ gradient via isomerization of retinal.

Summary Table: Key Complexes and Functions

Complex/Carrier	Main Function	Proton Pumping
Complex I	NADH to Q	Yes (4 H+)
Complex II	Succinate to Q	No
Complex III	QH2 to Cyt c	Yes (4 H+)
Complex IV	Cyt c to O2	Yes (2 H+)
ATP Synthase	H+ gradient to ATP	Utilizes gradient
Ubiquinone	Mobile electron carrier	No
Cytochromes	Electron transfer via heme	No
Iron-Sulfur Proteins	Electron transfer via Fe-S clusters	No

Example: ATP Yield from NADH

Each NADH molecule can result in the synthesis of approximately 2.5 ATP molecules via oxidative phosphorylation, due to the number of protons pumped and the stoichiometry of ATP synthase.