Cytochrome c, cytochrome b 5 and electron transfer

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1 Cytochrome c, cytochrome b 5 and electron transfer

2 Covalent bonds Cytochrome c is a major player in membrane associated electron transport systems in bacteria and mitochondria.

3 Cytochrome c has two axial ligands to the heme iron complex. Cytochrome c does not have the vacant pocket to receive O 2 or CO.

4 Cytochrome c has no O 2 (CO, NO) binding pocket, different from myoglobin and hemoglobin. Nat. Chem. Biol. 1, 223 (2005). Cytochrome c acts as a cardiolipin oxygenase required for release of proapoptotic factors. Programmed death (apoptosis) is turned on in damaged or unwanted cells to secure their clean and safe self-elimination. The initial apoptotic events are coordinated in mitochondria, whereby several proapoptotic factors, including cytochrome c, are released into the cytosol to trigger caspase cascades. The release mechanisms include interactions of B- cell/lymphoma 2 family proteins with a mitochondria-specific phospholipid, cardiolipin, to cause permeabilization of the outer mitochondrial membrane. Using oxidative lipidomics, we showed that cardiolipin is the only phospholipid in mitochondria that undergoes early oxidation during apoptosis. The oxidation is catalyzed by a cardiolipin-specific peroxidase activity of cardiolipin-bound cytochrome c. In a previously undescribed step in apoptosis, we showed that oxidized cardiolipin is required for the release of proapoptotic factors. These results provide insight into the role of reactive oxygen species in triggering the cell-death pathway and describe an early role for cytochrome c before caspase

5 Ubiquinone and the Proton Pump Ubiquinone is a lipid soluble cofactor that accepts and donates electrons in oxidationreduction reactions. These are reactions in which electrons are transferred from one molecule (oxidation) and accepted by another (reduction). Quinones play a role in pumping proteins across a membrane in order to create a proton gradient that's used to make ATP. Note that when two electrons are taken up, two protons (H + ) are added to neutralize the negative charge. In the reverse reaction (ubiquinol to ubiquinone: bottom to top) two protons are released when the electrons are given up.

6 Complex I Complex III Complex IV The red line traces the path of electrons released from a molecule called NADH. The electrons pass through three different membrane complexes called complex I, complex III, and complex IV. At each step, protons are pumped across the membrane. In complex IV the electrons are passed to oxygen (O 2 ) to make water. This final step is why you need oxygen to live.

7 Compound III

8 Compound III

Cytochrome c is a small heme protein which is associated with the inner

9 Cytochrome c plays a key part in electron transport associated with aerobic cellular respiration. Cytochrome c is a small heme protein which is associated with the inner membrane of the mitochondria. In the electron transport process it transfers electrons between Complex III and Complex IV.

10 Cytochrome c oxidase or Complex IV is a large transmembrane protein complex Found in bacteria and mitochondria. 4 Fe 2+ -cytochrome c + 8 H + in + O 2 4 Fe 3+ -cytochrome c + 2 H 2 O + 4 H + out

11 Subunits I and II of Complex IV excluding all other subunits.

Two electrons are passed from two cytochrome c's, through the Cu A and cytochrome a sites to the cytochrome a 3 - Cu B binuclear center, reducing the metals to the Fe +2 form and Cu +1.

12 Two electrons are passed from two cytochrome c's, through the Cu A and cytochrome a sites to the cytochrome a 3 - Cu B binuclear center, reducing the metals to the Fe +2 form and Cu +1. The hydroxide ligand is protonated and lost as water, creating a void between the metals that is filled by O 2. The oxygen is rapidly reduced, with two electrons coming from the Fe +2 cytochrome a 3, which is converted to the ferryl oxo form (Fe +4 =O). The oxygen atom close to Cu B picks up one electron from Cu +1, and a second electron and a proton from the hydroxyl of Tyr(244), which becomes a tyrosyl radical: The...

13 A cytochrome complex plays a key part in electron transport associated with the membranes of the thylakoids in the process of photosynthesis. It accepts electrons from Photosystem II through plastoquinone and contributes to proton transport across the membrane.

14 Cytochrome c has no O 2 (CO, NO) binding pocket, different from myoglobin and hemoglobin. Nat. Chem. Biol. 1, 223 (2005). Cytochrome c acts as a cardiolipin oxygenase required for release of proapoptotic factors. Programmed death (apoptosis) is turned on in damaged or unwanted cells to secure their clean and safe self-elimination. The initial apoptotic events are coordinated in mitochondria, whereby several proapoptotic factors, including cytochrome c, are released into the cytosol to trigger caspase cascades. The release mechanisms include interactions of B- cell/lymphoma 2 family proteins with a mitochondria-specific phospholipid, cardiolipin, to cause permeabilization of the outer mitochondrial membrane. Using oxidative lipidomics, we showed that cardiolipin is the only phospholipid in mitochondria that undergoes early oxidation during apoptosis. The oxidation is catalyzed by a cardiolipin-specific peroxidase activity of cardiolipin-bound cytochrome c. In a previously undescribed step in apoptosis, we showed that oxidized cardiolipin is required for the release of proapoptotic factors. These results provide insight into the role of reactive oxygen species in triggering the cell-death pathway and describe an early role for cytochrome c before caspase

15 Apoptosis the programmed death of a cell

16 Nature Rev. Immu. 2, 527 (2002) Schematic diagram of death receptor (DR) and mitochondrial pathways for the induction of apoptosis. Apoptosis can be induced by DR ligation (for example, FAS FASL) or by the disruption of mitochondrial integrity, such as occurs after DNA damage by cytotoxic agents or UV irradiation. DR-induced caspase activation is suppressed by FLIP, which interferes with caspase-8 activation. Induction of the DR pathway, which results in the activation of caspase-8, might lead directly to the activation of caspase-3, without requiring mitochondrial damage, or it might proceed though BID, resulting in the loss of mitochondrial transmembrane potential and the release of cytochrome c into the cytoplasm. Cytochrome c, in the presence of APAF1 and ATP, activates caspase-9, which results in the activation of caspase-3. The antiapoptotic molecules Bcl-2 and Bcl-X L protect against the loss of mitochondrial transmembrane potential that is induced by pro-apoptotic molecules, such as BAX and BAK. AIF, apoptosis-inducing factor; APAF1, apoptotic protease-activating factor 1; BAK, Bcl-2-antagonist/killer; BAX, Bcl-2-associated X protein; BID, BH3-interacting death-domain agonist; FADD, FAS-associated deathdomain protein; FASL, FAS ligand; FLIP, FADD-like IL-1- converting enzyme-inhibitory protein; MCL1, myeloid-cell leukaemia sequence 1; SMAC, second mitochondriaderived activator of caspase.

17 Cytochrome c is involved in apoptosis More recently, cytochrome c has been identified as an important mediator in apoptotic pathways. The release of mitochondrial cytochrome c into the cytoplasm stimulates apoptosis and is commonly used as an indicator of the apoptotic process in the cell. Serum cytochrome c levels may be an indicator of therapy-induced cell death burden. Under proapoptotic conditions, two Bcl-2 family proteins, Bax and Bak associate with the voltage-dependent anion channel component of the permeability transition (PT) pores on the outer membrane of the mitochondria. This calcium-dependent process allows the release of cytochrome c from the intermembrane space of the mitochondria into the cytoplasm. Cytochrome c also participates in the cytosolic caspase proteolytic cascade of apoptosis as a component of the apoptotic protease activating factor (Apaf). The association of cytochrome c with Apaf-1 results in the formation of the apoptosome protein complex which can recruit and activate pro-caspase 9 (Apaf-3). Activation of caspase 9 then facilitates the downstream activation of caspases 3 and 7 resulting in apoptosis. The cytochrome c- mediated release of calcium from the ER helps to initiate the apoptotic cascade since activation of both caspase 9 and caspase 3 is calcium-dependent.

Nature Struc. Biol. 10, 983 (2003). Figure 1. A representative signaling cascade of the mitochondriamediated apoptosis. X-ray irradiation causes doublestrand DNA breaks.

2 translocates from the nucleus to the mitochondria, where it activates Bak to release cytochrome c and other pro-apoptotic proteins such as Smac/DIABLO.

18 Nature Struc. Biol. 10, 983 (2003). Figure 1. A representative signaling cascade of the mitochondriamediated apoptosis. X-ray irradiation causes doublestrand DNA breaks. Via an unknown mechanism, the linker histone H1.2 translocates from the nucleus to the mitochondria, where it activates Bak to release cytochrome c and other pro-apoptotic proteins such as Smac/DIABLO. Cytochrome c induces the formation of apoptosome and subsequent activation of caspase-9 whereas Smac/DIABLO removes IAPmediated inhibition of caspases. In this diagram, the signaling steps prior to and after mitochondria are colored blue and orange, respectively. Although not shown, the antiapoptotic Bcl-2 and Bcl-xL also reside in the outer membrane of mitochondria.

19 PNAS, 102, (2005). Model of apoptosome formation. Apaf-1 is associated with datp. Upon cytochrome c binding, Apaf- 1 hydrolyzes datp. If there is extra datp/atp, dadp is exchanged with datp and Apaf-1 forms the active apoptosome. When Apaf-1 is incubated with cytochrome c without extra datp/atp, dadpbound Apaf-1 forms the inactive aggregate.

20 AAPS Journal 8, E277 (2006). Increasing evidence suggests that ROS play a key role in promoting cytochrome c release from mitochondria. Cytochrome c is normally bound to the inner mitochondrial membrane by association with cardiolipin.3 Peroxidation of cardiolipin leads to dissociation of cytochrome c and its release through the outer mitochondrial membrane into the cytosol. The mechanism by which cytochrome c is released through the outer membrane is not clear. One mechanism may involve mitochondrial permeability transition (MPT), with swelling of the mitochondrial matrix and rupture of the outer membrane (Figure 3). ROS may promote MPT by causing oxidation of thiol groups on the adenine nucleotide translocator, which is believed to form part of the MPT pore. Cytochrome c release may also occur via MPT-independent mechanisms and may involve an oligomeric form of Bax6 (Figure 3). Cytochrome c in the cytoplasm triggers the activation of caspase-9, which triggers the caspase cascade and ultimately leads to apoptosis. Figure 3. Cytochrome c release from mitochondria. Cytochrome c ( ) is normally associated with cardiolipin on the inner mitochondrial membrane. Cytochrome c is dissociated upon oxidation of cardiolipin and is believed to be released out of mitochondria either by mitochondrial permeability transition resulting in mitochondrial swelling and rupture of the outer membrane, or by channels formed by oligomerization of Bax. In the cytoplasm, cytochrome c activates caspase-9 and promotes apoptosis.

21 1. Interaction with cadiolipin dissociates the axial ligand of cytochrome c. 2. Oxygen binding site is generated by cadiolipin. 3. Cytochrome c interacts with H 2 O 2 (or generated O 2 -. and/or H 2 O 2 ).

22 Peroxidase ROOR + electron donor (2 e - ) + 2H + ROH + R OH Cytochrome c peroxidase (CCP) CCP + H 2 O ferrocytochrome c + 2H + CCP + 2H 2 O + 2 ferricytochrome c Peroxidase needs the open pocket on the heme binding site to carry out the reaction. This pocket of cytochrome c is generated by the interaction with cadiolipin

23 Science 292, 727 (2001) Proapoptotic BAX and BAK: A Requisite Gateway to Mitochondrial Dysfunction and Death Multiple death signals influence mitochondria during apoptosis, yet the critical initiating event for mitochondrial dysfunction in vivo has been unclear. tbid, the caspase-activated form of a BH3- domain only BCL-2 family member, triggers the homooligomerization of multidomain conserved proapoptotic family members BAK or BAX, resulting in the release of cytochrome c from mitochondria. We find that cells lacking both Bax and Bak, but not cells lacking only one of these components, are completely resistant to tbid-induced cytochrome c release and apoptosis. Moreover, doubly deficient cells are resistant to multiple apoptotic stimuli that act through disruption of mitochondrial function: staurosporine, ultraviolet radiation, growth factor deprivation, etoposide, and the endoplasmic reticulum stress stimuli thapsigargin and tunicamycin. Thus, activation of a multidomain proapoptotic member, BAX or BAK, appears to be an essential gateway to mitochondrial dysfunction required for cell death in response to diverse stimuli. Cell Metab. 1, 393 (2005) Mitochondrial dysfunction resulting from loss of cytochrome c impairs cellular oxygen sensing and hypoxic HIF-α activation While cellular responses to low oxygen (O 2 ) or hypoxia have been studied extensively, the precise identity of mammalian cellular O 2 sensors remains controversial. Using murine embryonic cells lacking cytochrome c, and therefore mitochondrial activity, we show that mitochondrial reactive oxygen species (mtros) are essential for proper O 2 sensing and subsequent HIF-1α and HIF-2α stabilization at 1.5% O 2. In the absence of this signal, HIF-α subunits continue to be degraded. Furthermore, exogenous treatment with H 2 O 2 or severe O 2 deprivation is sufficient to stabilize HIF-α even in the absence of cytochrome c and functional mitochondria. These results provide genetic evidence indicating that mtros act upstream of prolyl hydroxylases in regulating HIF-1α and HIF-2α in this O 2 sensing pathway.

24 Am. J. Physiol. Hear Circ. Physiol. 282, H726 (2002) Cardiac dysfunction in mice lacking cytochrome-c oxidase subunit VIa Ann. Neurol. 17, 414 (1985) Fatal infantile mitochondrial myopathy and renal dysfunction caused by cytochrome c oxidase deficiency: Immunological studies in a new patient Genetic 176, 937 (2007) Mutations in Cytochrome c Oxidase Subunit VIa Cause Neurodegeneration and Motor Dysfunction in Drosophila J. Mol. Cell Cardiol. 35, 357 (2003) Inhibition of mitochondrial permeability transition prevents mitochondrial dysfunction, cytochrome c release and apoptosis induced by heart ischemia. Biochim. Biophys. Acta 1807, 1336 (2011) Functional effects of mutations in cytochrome c oxidase related to prostate cancer. Proc. Nat. Acad. Sci., USA 106, 3402 (2009) A mitochondrial DNA mutation linked to colon cancer results in proton leaks in cytochrome c oxidase. J. Neurosci. 20, 5715 (2000) Delayed Mitochondrial Dysfunction in Excitotoxic Neuron Death: Cytochrome c Release and a Secondary Increase in Superoxide Production EMBO J. 20, 661 (2001) A reversible component of mitochondrial respiratory dysfunction in apoptosis can be rescued by exogenous cytochrome c

25 Cytochrome b5

26 Cytochromes b 5 are uniquitous electron transport hemoproteins found in animals, plants, fungi and purple phototropic bacteria. The microsomal and mitochondrial variants are membrane-bound, while bacterial and those from erythrocytes and other animal tissues are water-soluble. Cytochrome b 5 reductase NADH + H ferricytochrome b 5 = NAD ferrocytochrome b 5 L-ascorbate + ferricytochrome b 5 = monodehydroascorbate + ferrocytochrome b 5 Defects in CYB5A are the cause of methemoglobinemia CYB5A-related (METHB-CYB5A). A form of methemoglobinemia, a hematologic disease characterized by the presence of excessive amounts of methemoglobin in blood cells, resulting in decreased oxygen carrying capacity of the blood, cyanosis and hypoxia. Fe(II) binds to O 2. Fe(III) does not bind to O 2.

27 Rat cytochrome b 5 Human cytochrome b 5

28 Cytochrome P450 28

30 Cytochrome b 5 Cytochrome b5 reductase 30

Fig. 1. The elements of redox chains which are entered into the twomembrane picture. Inner membrane contains respiratory chain, namely complexes I, II, III and IV as well as ATP-synthase (complex V).

31 Fig. 1. The elements of redox chains which are entered into the twomembrane picture. Inner membrane contains respiratory chain, namely complexes I, II, III and IV as well as ATP-synthase (complex V). It contains adenine nucleotide translocator (ANT) also. Outer membrane contains cytochrome b5 reductase and cytochrome b5. Intermembrane transfer of an electron from NADH to cardiolipin (LOO) via cytochrome b5 reductase cytochrome b5 cytochrome c initiates the formation of anion LOO. Aging Res. Rev. 9, 200 (2010)

Biophysics 490M Project

Biophysics 490M Project Dan Han Department of Biochemistry Structure Exploration of aa 3 -type Cytochrome c Oxidase from Rhodobacter sphaeroides I. Introduction: All organisms need energy to live. They