OXIDATIVE PHOSPHORYLATION

Modified from MW King Mitochondrialny łańcuch oddechowy umiejscowienie: wewnętrzna błona mitochondrialna, skład > 80 białek zorganizowanych w 5 kompleksów enzymatycznych (I-V) Electron shuttle molecules: Coenzyme Q (CoQ); Cytochrome c (Cyt c) Funkcje ogólne: Produkcja ATP z procesów redukcji tlenu w celu produkcji energii dla funkcji komórkowych. Drogi: Pobieranie elektronów generowanych dzięki ich donorom w bezpośrenich procesach metabolicznych, Elektrony są sekwencyjnie przenoszone przez grupy redox na końcowy ich acceptor, którym jest tlen. Wolna energia : zużywana do pompowania protonów z macierzy mitochondrialnej do przestrzeni międzybłonowej. Wytwarza się elektrochemiczny gradient w poprzek wewnętrznej błony mitochondrialnej, Protein flux back into mitochondrial matrix through Complex V is coupled to ATP synthesis See: Animated diagram of proton pump & ATP synthesis Other mitochondrial functions Beta oksydacja wolnych kwasów tłuszczowych, Biosynteza: pirimidyn; aminokwasów; fosfolipidów; nukleotydów; hemu

MITOCHONDRIAL RESPIRATORY CHAIN: COMPOSITION
Complex	I	II	III	IV	V
Enzyme	NADH-CoQ Reduktaza	Sukcynylo-CoQ Reduktaza	CoQ-Cytochrome C Reductase	Cytochrome C Oxidase	ATP Synthase
Inhibitor	Rotenone Amytal	TTFA malonate	Antimycin A	Cyanide Carbon Monoxide Azide	Oligomycin
Nuclear DNA Subunits	39	4	10	10	~14
Mitochondrial DNA Subunits	7 ND1-6, ND4L	0	1 Cytochrome b	3 Cytochrome oxidase I , II , III	2 ATPase 6 ATPase 8
Flavoprotein	Flavin mononucleotide	FAD (SDHA)
Iron protein	Siarczek żelaza (FeS) protein NDUFS1	FeS protein SDHB	Rieske FeS ; Cytochrome (Heme) b, c1	Cytochrome (Heme) a, a3
Other features		Membrane proteins Cytochrome b560 SDHC SDHD		Copper protein
Disorders	General Alpers Alzheimer's/   Parkinsonism Cardiomyopathy Deficiency   Barth   Lethal Infantile Encephalopathy Infantile CNS Leber's Leigh Longevity MELAS MERRF Myopathy ± CNS PEO Spinal cord	General Kearns-Sayre Leigh's Myopathy   Infantile   ± CNS Paraganglioma Pheochromocytoma	General Cardiomyopathy Fatal infantile GRACILE Leber's Myopathy   ± CNS PEO	General Alper's Ataxia Deafness Leber's Leigh's Myopathy   Infantile     Fatal     Benign   Adult   Rhabdomyolysis PEO, KSS, MNGIE MERRF, MELAS	ATP12 Cardiomyopathy Leber's Leigh NARP
Notes Większość encefalomiopatii mitochondrialnych jest związana z mutacjami mutacjami w mt DNA Nuclear mutations ® Defects in respiratory chain Leigh's: Flavoprotein subunit of succinate dehydrogenase (complex II) : Fatal multisystemic complex I deficiency: Infantile onset NADH Dehydrogenase (ubiquinone) Fe-S Protein 4 18-kD (AQDQ) subunit of complex I Barth syndrome: Tafazzins Hereditary spastic paraparesis 7: Paraplegin ~ 1,000 mitochondrial peptides not involved in the respiratory chain: Encoded by nuclear DNA Superoxide dismutase 2 (SOD2) Intramitochondrial free radical scavenging enzyme Degrades superoxide produced as a byproduct of oxidative phosphorylation

COMPLEX I (NADH-Ubiquinone Oxidoreductase): Features & Deficiency¹

• Kompleks I: budowa

• Skład

• Największy kompleks łańcucha oddechowego

• 46 białek

• 7 kodowanych mitochondrialnie białek

• 39 kodowanych przez nDNA.

• Główne podjednostki : flawoproteina; Iron-sulfur protein; Hydrophobic fraction

• Subunits involved in Complex I structure

• NDUFAB1 (SDAP) : Carrier of fatty acid chain

• NDUFA1 (MWFE)

• Primarily expressed in heart & skeletal muscle

• Disorders: Encephalopathies

• Subunits involved in regulation of Complex I activity

• NDUFS4 (AQDQ)

• Functions: Increased Complex I activity with phosphorylation

• Disorders: Multisystem childhood encephalopathy with Complex I deficiency; Leigh syndrome

• Protein involved in Complex I assembly

• MYC-induced mitochondrial protein (B17.2L; Mimitin)

• Disorder: Childhood encephalopathy

• External link: Components

• General structure

• L-shaped

• Hydrophobic arm

• Contains mtDNA-encoded subunits

• Attaches complex to inner mitochondrial membrane: Embedded in membrane

• ? Formed by 2 small & large subcomplexes (in Neurospora)

• Role in assembly played by: CIA30; CIA84

• Hydrophilic peripheral arm

• Flavoprotein

• Contains NADH binding site & redox active centers

• Extends into mitochondrial matrix

• Iron�sulphur protein fraction

• Facilitates further electron transport to hydrophobic fraction

• Location: Parts in

• Mitochondrial inner membrane

• Mitochondrial matrix

• Complex I: Associations (Respirasome)

• Supercomplex of I, III, IV

• Stabilizes structure of Complex I

• Stoichiometry of I:IIII:IV = 1:2:4

• Deficiency in other Complexes may produce loss of Complex I function

• Complex I: Functions

• Transport of electrons from NADH to ubiquinone

• Electron source: NADH

• Co-factor: Flavin mononucleotide

• Transport: via eight redox groups, iron�sulphur clusters

• Electron acceptor: Ubiquinone

• Ubiquinone function: Transfers of electrons to next complex in the chain (Complex III)

• Simultaneous shunting of protons

• Out of mitochondrial matrix

• Across inner mitochondrial membrane

• Into intermembrane space

• Stoichiometry: 4H⁺/2e^-

• Disorders

• General

• Lactate levels & muscle morphology may be normal

• Most common clinical phenotype in childhood: Leigh syndrome

• Most common cause of Complex I deficiency: Nuclear gene mutations

• Complex I ± Other complexes deficient

• Alper's

• Alzheimer's/Parkinsonism

• Cardiomyopathy

• Deficiency: Barth; Infantile CNS

• Encephalopathy, Adult onset

• Leber's (LHON)

• Leigh

• Longevity

• MELAS

• MERRF

• Myopathy

• ± CNS

• PEO

• Spinal cord

• Isolated Complex I deficit: mtDNA mutations

• MELAS: tRNA^Leu (A3243G & T3271C)

• Myopathy: tRNA^Leu (T3250C, A3251G & A3303G)

• Leber's (LHON): tRNA^Lys (G11778A), ND1 (G3460A) & ND6 (T14484A)

• MERRF: tRNA^Lys (G8363A), ND1 (A8344G), tRNA^{Ser (MTTS2)}

• Leigh syndrome: ND4 (G14459A)

• Long QT syndrome: tRNA^leu (T3394C)

• Diabetes & Fanconi syndrome: Multiple deletion

• Ataxia, Hypogonadism & Choroidal dystrophy: Multiple deletion

• Isolated Complex I deficit: Nuclear mutations

• Clinical features: General

• Nuclear mutations more common than mitochondrial

• Onset: Childhood; Mean = 4 to 5 months

• Male:Female = 3.5:1

• Prognosis: Death in 1st 2 years in 67%; ? Better with normal blood lactate

• Lactic acidosis: Blood 85%; CSF 90%

• Inheritance: Usually autosomal recessive

• MRI: Abnormal

• Specyficzne zespoły

• Ciężka noworodkowa kwasica mleczanowa,

• Zespół Leigha: NDUFS3; NDUFS4; NDUFS7; NDUFS8

• Makrocefalia z postępującą leukodystrofią: NDUFV1

• Noworodkowa kardiomiopatia z kwasicą mleczanową

• encefalomiopatia: NDUFV1

• Hepatomegalia z kanałopatią nerkową

• Lethal neonatal: NDUFS6

• Cardiomyopathy + Encephalomyopathy: NDUFS2; NDUFV2

• Childhood encephalopathy: NDUFS1; MYC-induced mitochondrial protein; NDUFA1

COMPLEX II (Succinate Dehydrogenase-CoQ Oxoreductase): Features & Deficiency

• Functions

• Mitochondrial respiratory chain

• Catalyzes oxidation of succinate to fumarate

• Transfers electrons to ubiquinone pool of respiratory chain

• Krebs cycle

• Composition

• Subunits: General

• Number: 4

• All nuclear encoded

• Subunits A & B form succinate dehydrogenase (SDH)

• Subunits C & D anchor enzyme to membrane

• Flavoprotein: FAD (SDHA; Fp)

• Functions: Catalytic site; Covalently bound FAD cofactor

• Mutations

• Leigh syndrome with Complex II deficiency

• Late onset neurodegenerative disorder

• Iron-Sulfur protein: SDHB (Ip)

• Function: Electron transfer between FAD and membrane-bound quinone

• Structure: Contains three different iron-sulphur clusters

• SDHB mutations

• Reduced tumor suppression

• Neoplasms: Pheochromocytoma & Paraganglioma

• Cytochrome b subunits: SDHC ; SDHD

• Integral membrane proteins: Bind Fp & Ip to matrix

• SDHC & SDHD mutations: Paraganglioma

• Location of Complex II

• Matrix side of mitochondrial inner membrane

• Binding to membrane is dependent on 2 small (15.5 and 13.5 kDa) proteins SDHC & SDHD

COMPLEX III (CYTOCHROME REDUCTASE): Features & Deficiency

Complex III Features

• Composition

• Nuclear subunits

• Number: 10

• Components

• Cytochrome c1 (CYC1)

• Ubiquinone-binding protein (UQPC)

• Ubiquinol-cytochrome c reductase core protein II (UQCRC2)

• Rieske FeS protein

• Mitochondrial subunits

• Number: 1

• Component: Cytochrome b

• Location of Complex III: Inner mitochondrial membrane

• Associations

• Supercomplex of Complexes I, III, IV

• Association of Complexes III & IV may be stabilized by cardiolipin

• Defects in cardiolipin remodeling: Barth

Complex III Functions

• Transfers electrons from ubiquinol to cytochrome c

• Coupled with transfer of electrons across inner mitochondrial membrane

• Contains 3 redox centers

• Cytochrome b

• Cytochrome c

• Rieske FeS protein

Complex III disorders

• Nie ma zidentyfikowanej mutacji w podjednostce kodowanej jądrowo

• Selektywne biochemiczne defekty kompleksu III

• Cytochrome b : Mitochondrial; Sporadic

• Leber's

• Myopathy ± CNS

• GRACILE

• UQCRB

• Other defects

• Cardiomyopathy

• Fatal infantile

• PEO

• Tubulopathy, encephalopathy & liver failure

COMPLEX IV (CYTOCHROME OXIDASE): Features & Deficiency

Complex IV Composition & Related Proteins

• Nuclear subunits

• Number: 10

• Presumed to play a structural & regulatory role in COX

• Mitochondrial subunits

• Number: 3

• Largest COX subunits

• Form catalytic core of COX

• Contain the 3 copper atoms & 2 heme A molecules

• Serve as prosthetic groups in holoenzyme

• Directly involved in electron transfer

• Assembly factors related to COX

• Essential components of COX biosynthetic pathway

• Expression of individual COX subunits

• Provide prosthetic groups to the apoenzyme

• Factors include: SURF-1; SCO1; SCO2; COX 10; COX 17

Nuclear mutations

• General

• 4 mutations in proteins required for assembly of holocomplex

• 1 mutation in mRNA binding protein: LRPPRC

• No mutations in nuclear encoded structural proteins of COX

• Specific mutations

• Surfeit-1 (SURF-1) : Leigh syndrome (French-Canadian type)
  l Chromosome 9q34; Recessive

• COX subunits reduced: All except Va & Vb

• SCO1 : Neonatal hepatic failure & Ketoacidotic coma²
  l Chromosome 17p13.1; Recessive

• SCO2 : Cardioencephalomyopathy, fatal infantile
  l Chromosome 22q13; Recessive

• COX subunits reduced: I & II

• COX10 (Heme A:farnesyltransferase) : Ataxia; Encephalopathy
  l Chromosome 17p13.1-q11.1; Recessive

• COX subunits reduced: II

• LRPPRC: Leigh syndrome; Metabolic acidosis
  l Chromosome 2q16; Recessive

Mitochondrial mutations

• COX I subunit

• Leber optic atrophy

• Sideroblastic anemia, acquired idiopathic

• Encephalopathy with hearing loss

• Colorectal cancer

• Exercise intolerance & Myoglobinuria

• COX II subunit

• Ataxia

• Myopathy ± CNS

• COX III subunit

• Leber optic atrophy

• Myoglobinuria

• Exercise intolerance

• Encephalopathy

• Spastic paraparesis, Mental retardation & Ophthalmoplegia

Other syndromes associated with Complex IV deficiency

• Alexander disease

• Alper's

• Ataxia

• Copper deficiency

• Deafness

• Hyperhomocysteinemia

• Leber's

• Leigh's

• Myopathy

• Infantile-onset

• Fatal

• Benign

• Adult-onset

• Rhabdomyolysis

• PEO

• KSS

• MNGIE

• MERRF

• MELAS

COMPLEX V (ATP Synthase): Features & Deficiency

• Composition

• mtDNA encoded: 2 subunits

• Nuclear DNA encoded: 14 subunits

• Integral membrane component: F0

• Composed of at least seven subunits

• mtDNA encodes 2 subunits: ATPase 6 & 8

• Peripheral moiety: F1

• Composed of six subunits

• Nuclear DNA encodes all subunits

• Uses proton gradient to convert ADP to ATP

• Function: Couples proton gradient generated by respiratory chain to ATP synthesis

• Proton flow from intermembrane space to matrix

• Conversion of ADP + inorganic phosphate to ATP

• Mutations

• mtDNA for ATPase 6

• Leigh (MILS): High levels (> 90%) of T8993G or T8993C mutations in brain & blood

• NARP: Intermediate levels (70% to 90%) of T8993G or T8993C mutations in brain & blood

• Bilateral striatal necrosis: T9176C mutation

• Nuclear encoded protein mutations

• ATP12

• Assembly gene

• Congenital multisystem disorder

• Cardiomyopathy

• No mutations identified in nuclear encoded subunits

Combined deficiencies of Complexes I, III, IV, and V

• Causes: Mutations

• mtDNA

• Components of mitochondrial translation apparatus encoded by nuclear genes

• Synthesis of 13 mitochondrial gene-encoded proteins

• Occurs on a dedicated mitochondrial translation apparatus

• Requires tRNAs and ribosomal RNAs (rRNAs) encoded in mtDNA

• Other factors

• Translation factors

• Mitochondrial ribosomal (mitoribosome) proteins: All encoded by nuclear genes

• Mammalian initiation factors: IF2 & IF3

• Elongation factors: EFTu, EFTs, EFG1 & EFG2

• Release factor: RF1

• Ribosomal recycling factor: RRF

• Mitoribosomes

• 55S particles

• Composed of small (28S) & large (39S) subunits

• Contain a much higher protein:RNA ratio than bacterial 70S ribosome

• Disorders

• mtDNA depletion

• Thymidine kinase 2 (TK2)

• Other nuclear gene mutations

• EFG1: Hepatoencephalopathy

• MRPS16: Protein of small ribosomal subunit

• EFTs (TSFM)

• Fatal mitochondrial encephalomyopathy

• Fatal hypertrophic cardiomyopathy

• ANT1: Cardiomyopathy

Lactate metabolism⁴

• Production

• Pathways

• Glycogenolysis

• Glycolysis

• Increased production with

• Greater exercise power output

• Increased oxygen uptake

• Produced even under fully aerobic conditions

• Shuttles

• Molecules: Lactate exchange facilitators

• Membrane transporters: Monocarboxylate transport proteins (MCTs)

• Stereo-selective for L-(+)-lactate

• pH dependent

• Sarcolemmal lactate transporters may have role in regulation of intracellular pH

• Lactate transport in skeletal muscle: MCT1 (SLC16A1) & MCT4 (SLC16A3)

• Inactivity: Reduces MCT1 & MCT4 expression

• Training: Increases MCT1 & MCT4 expression

• Brief, high intensity exercise: Reduces MCT1 & MCT4 expression

• Scaffolding proteins

• Cell-cell lactate exchanges between

• White-glycolytic & red-oxidative fibres in a working muscle bed

• Working skeletal muscle & heart

• Tissues of net lactate release & gluconeogenesis

• Astrocytes & neurons in brain: Linked to glutamatergic signalling

• Intracellular lactate exchanges

• Mitochondrial lactate oxidation complex

• Proteins involved

• Mitochondrial lactate/pyruvate transporters (MCT)

• Scaffolding glycoprotein basigin (CD147) : Chaperone protein

• Lactate dehydrogenase

• Cytochrome oxidase

• Location: Mitochondrial reticulum

• Function

• Proteins work in conjunction to permit lactate oxidation in actively respiring cells

• Establishes concetration & proton gradients

• Needed by cells with high mitochondrial densities: Cardiac & Skeletal muscle

• Sarcolemma

• Protein: Sarcolemmal lactate/pyruvate transporter MCT1 (SLC16A1)

• Training effects: Change expression of related proteins

• Disposal

• Pathways

• Oxidative metabolism

• Rest: 50%

• Exercise: 75%

• Cori cycle

• Contribution: 25% during exercise

• Pathway: Conversion of lactate to glucose (gluconeogenesis)

• Increased disposal with

• Endurance training: Reduces arterial lactate

• Disorders

• SLC16A1 Lactate transporter defect

• Epidemiology: 2 patients

• Genetics: Heterozygous missense mutations; Lys204Glu, Gly472Arg

• SLC16A1 protein

• Transporter defect location: Erythrocytes & Muscle

• Clinical: Pain after exercise

• Lab: Erythrocyte lactate clearance rates decreased to 40% to 50% of normal control

• Lactate dehydrogenase, M (muscle) subunit

• Basigin : Blood group OK