16 Respiratory Assemblies

Dr. Chirantan Rawal

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Respiratory Assemblies

 

Objectives

  1. To study respiratory enzyme as free floating v/s super complex theory.
  2. To understand the assembly of respiratory proteins.
  3. To understand the role of assembly factors in assembly of supercomplexes.

   

Introduction

  • Mitochondria generate ATP through oxidative phosphorylation in eukaryotic microorganisms.
  • Oxidative Phosphorylation is the process whereby the free energy that is released when electrons are transferred along the electron transport (respiratory) chain is coupled to the formation of adenosine triphosphate (ATP) from adenosine diphosphate (ADP) and inorganic phosphate (Pi).
  • In intact mitochondria and in special preparations of sub mitochondrial particles, the transport of electrons and the phosphorylation of ADP are tightly coupled reactions.
  • In damaged mitochondria, respiration (i.e., electron transport) may occur unaccompanied by oxidative phosphorylation. When this happens the mitochondria are said to be uncoupled.
  • Oxidative phosphorylation is the main source of energy in aerobic cells.
  • In uncoupled mitochondria, because electron transport may still occur, free energy may still be released as the electrons are transferred down the transport chain. However, this energy is not trapped as ATP and appears instead as heat.
  • Mitochondria play an important role in satisfying the requirement of cellular energy through oxidative phosphorylation. Apart from this it plays many other important role which are…

     It buffers the cytoplasmic calcium

 It creates and liberate reactive oxygen

 It liberates metabolites that control crucial pathways such as succinate and α-ketoglutarate

 It helps in controlling apoptosis

 It assists the cell to adopt change in substrate availability by signaling pathways

 They modify their organization and dynamics for quality control.

  • So role of mitochondria indicates that it plays a vital task in maintaining the balance with in the cell. It requires a strong regulation. Regulation of mitochondrial activity through expression, transcription and translation.
  • Recent studies indicate that regulation of mitochondrial activity is also achieved by dynamic assembly/ organisation of the respiratory protein complexes in the inner mitochondrial membrane.
  • The mitochondrial electron transport chain i.e. mitochondrial respiratory chain consist of five protein complexes:

    Complex I : NADH-ubiquinone oxidoreductase

Complex II : succinate-ubiquinone oxidoreductase

Complex III: Ubiquinone cytochrome- c oxidoreductase

Complex IV: cytochrome-c oxidase

Complex V: ATP synthase

  • Electrons are released from NADH2 enters in to Complex I. Then electron is transferred through a series of electron and hydrogen carrier i.e. complex II to V. This transfer of electron and proton generates the gradient. This gradient drives the synthesis of ATP through ATP synthase (Complex V).
  • The energy derived from the movement of these protons is used to synthesize ATP from ADP and phosphate. Formation of ATP by this mechanism is referred to as oxidative phosphorylation.

 

     Generation of ATP by Electron Transport

  • In a number of different mode of microbial metabolisms including respiration and photosynthesis, ATP is generated by transporting electron through the chain of carrier molecules with fixed orientation in a cell membrane.
  • Although the complexity and component of electron transport chain vary, they have a certain common features:
  • The component of chain are carrier molecules capable to undergoing reversible oxidation and reduction ; each member of chain is capable being reduced by reacting with the carrier molecules that precedes it and oxidized by the carrier that follows it.
  • In any specific example of an electron transport chain, certain members transport hydrogen atoms while other transport only electron.
  • The orientation of carrier in the cell membrane is such that hydrogen carriers transport in the direction toward the outside of the cell and electron carrier transport toward the inside.
  • Thus, at each conjunction in the chain of a hydrogen carrier and an electron carrier, a proton is transported out of the cell.
  • The cell membrane is otherwise impermeable to proton; as consequence electron transport traps a portion of the chemical energy released by the net reaction of the chain in the form of gradient across the membrane of proton and electric charge.
  • Such a gradient termed as proton motive force (Δp) is form of potential energy capable of doing work : it drives certain permease system that concentrate externally supplied substrate within the cell; it provides the energy for flagellar-mediated cell motility and its drives the energy requiring synthesis of ATP from ADP.
  • The synthesis of ATP at the expense of proton motive force is catalyzed by complex membrane bound enzyme, ATP phosphohydrolase (some time termed as ATPase ) composed in all bacterial studied , of two multi component protein BF0 and BF1.
  • The subunit composition and membrane insertion of BF0 and BF1 are shown in figure.
  • The α and β subunit of BF1are arranged alternately to form a hollow hexagon, the central hole of which contain the γ subunit associated with other subunit δ and ε.
  • Thus BF1 probably has the subunit structure α3β3γδε .
  • The α and β subunit form the catalytically active portion of structure ,the site where ATP is synthesized from ADP and inorganic phosphate ;the γ,δ and ε subunit form a proton translocating stalk and gate that bring to the active site at the proper rate the proton that drive reaction.
  • The peptide form a proton channel through the membrane they are hihly hydrophobic accounting for their intra membrane location
  • ATP phosphohydrolase catalyzed a reversible reaction .ATP can be synthesize at a expense of proton motive force ,or in certain case a proton motive force can be established at the expense of intracellular ATP.

 

 Traditional fluid model v/s super complex structure (Respirosomes)

  • There are two different views regarding the arrangement of the respiratory enzymes within the mitochondrial membranes.
  • The respiratory enzyme complexes were initially anticipated to be closely packed. Such packed structure assumes to provide more effectiveness in electron transport.However, this original model was gradually discarded and replaced by the fluid model
  • Fluid model: Initially it was believed that respiratory enzyme complex exist as free floating structures within the mitochondrial membranes. Over here it was assumed that cytochrome c ubiquinone acts as a connecting molecule. Respiratory enzymes are diffused within the inner mitochondrial membranes. In this model, the respiratory complexes are observed as free unit set in the inner membrane.
  • MRC model: (Mitochondrial Respiratory Complex model )
  • The mitochondrial electron transport chain i.e. mitochondrial respiratory chain consists of five protein complexes.
  • Due to advance in the accessibility of sensitive biochemical assays, mitochondrial respiratory complex model (MRC) is slowly replacing the traditional model i.e. floating complex in the inner mitochondrial membrane. The regular and frequent observations of respiratory complexes associating with each other to form super complexes has gradually change the idea of a ‘fluid state’ model of the ETC.
  • MRC model suggest that respiratory chain is composed of the stable super complexes. These super complexes are functional structures of respiration.
  • MRC model suggest that respiratory enzymes forms a solid complexes. This solid complex consists of different complexes which are connected with each other to form super complexes.

   

SUPER COMPLEXES ASSEMBLY

  • MRC model suggest that respiratory enzymes forms a solid complexes. This solid complex consists of different complexes which are connected with each other to form super complexes.
  • The most general super complexes recognized are Complex I/IIIn, Complex I/IIIn/IVn and Complex III/IVn
  • Complex II is observed to be free. In plant and mammalian mitochondria it is observed free. It is not associated with other complex. It is observed that small portion of the Complex II is associated with super complex I/III/IV.
  • Complex V observed as dimer. It migrates with other super complexes.
  • However, while lots of structural evidence was presented for the existence of supercomplexes. But less number of evidence was available to specify their functional implication.
  • It is observed that Complex I assembly is depended on the Complex III (Acin-Perez et al., 2004). It is also dependent on the Complex IV (Diaz et al., 2006; Li et al., 2007).
  • It is found that Complex I was unstable in the absence of Complex III. lack of Complex IV affects assembly of Complex I (Li et al., 2007).
  • The absence of Complex I does not affect either Complex III or Complex IV (Acin-Perez et al., 2004; Li et al., 2007).
  • Super complex formation provides better substrate channelling and transport of electron. It also provides better structural stability.
  • So Complex I has dependence on other complexes for their formation and stability. This leads to an hypothesis that formation of super complex assembly was compulsory for the stability of assembled, individual respiratory Complex I.
  • Experimental findings suggest that first individual complexes will form. This is followed by the formation of super complex. Mitochondrial DNA encoded products sequential incorporated in to the individual respiratory complexes. There is a small gap between formation of individually complexes and super complex assembly.
  • In Neurosopra sp., it is observed that assembly and synthesis of Complex I was very closely associated with super complex formation.
  • In mouse mitochondria observed that partial Complex I is formed first. Size of that complex is 830 KDa. Then Complex III and Complex IV associate with complex I subunit with the help of assembly factor. After attachment of complex III and Complex IV other subunit of Complex I i.e. NDUDS4 and NDUFV1 is attached to form a super complex assembly. This indicates that supercomplexes assembly precedes the assembly of individual complex formation. Stability of complex I may require the association of complex IV and III.
  • Assembly factors: These factors assist in the assembly of respiratory enzymes. These factors have significance in trying to study the assembly of these complexes. It has importance in understanding the diseases related to mitochondrial disorders. It is belived that these assembly factors help in assembling the supercomplexes after assembly of individual respiratory complex.
  • Cardiolipin: It is found in mitochondrial membrane. Low concentration of this factor leads to reduced membrane potential, ATP synthesis and overall mitochondrial function. Cardiolipin attach to Complex I, Complex III, Complex IV and Complex V in the inner mitochondrial membrane. It has six binding sites for complex III and IV.
  • Rcf-1 and Rcf-2: It is coded by genes Hig-1. Their function is not known to us. These factors are present in the mitochondrial inner membrane. Lack of these factors causes mitochondrial dysfunction. From studies it was revealed that they are playing crucial role in the formation of supercomplexes. Their absence does not affect the individual complexes.
  • ATP/ADP carrier protein: These are the proteins found in the inner mitochondrial membrane. It is responsible for the transfer of ATP through the mitochondrial membrane. Absence of these carrier proteins have lethal effect on the yeast cells suggest its role more than the transporter.
  • Other putative super complex assembly factors: Prohibitins (PHB) phosphatidyl ethanolamine (PE).

 

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References

  • Rasika Vartak, Christina Ann-Marie Porras, Yidong Bai Respiratory supercomplexes: Structure, function and assembly. PROTEIN & CELL ( 2013)DOI: 10.1007/s13238-013-3032.
  • H. McBride, L. Scorrano, Mitochondrial dynamics and physiology, Biochim. Biophys. Acta (2013) 148–149.
  • Acin-Perez, R., Bayona-Bafaluy, M.P., Fernandez-Silva, P., Moreno-Loshuertos, R., Perez-Martos, A., Bruno, C., Moraes, C.T., and Enriquez, J.A. (2004). Respiratory complex III is required to maintain complex i in mammalian mitochondria. Mol Cell 13, 805–815.
  • Acín-Pérez, R., Fernández-Silva, P., Peleato, M.L., Pérez-Martos, A., and Enriquez, J.A. (2008). Respiratory active mitochondrial supercomplexes. Mol Cell 32, 529–539.

Books

  • Harper’sIllustrated Biochemistry by Robert K.. Murray, Daryl K. Granner, Peter A. Mayes, 26th Edition (2003)
  • Lehninger’S Principle of Biochemistry David L. Nelson and Michael M. Cox,5th Edition (2008)
  • Textbook of Biochemistry, 4th Edition Donald Voet, Judith G. Voet (2011)