Cytochrome C, Cytochrome B5 and Electron Transfer

Cytochrome c, cytochrome b5: electron transfer and new functions

1 Iron Protoporphyrin IX, heme b Heme a Prosthetic group Heme a is a form of heme found A flat and planar structure. In cytochromes a and a3. -. Itself Toxic O2 , H2O2 are formed. Insoluble 2 Covalent bonds

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

3 Myoglobin, Hemoglobin His E7 Distal side

Heme iron

His F8 Proximal side

Myoglobin has a strong affinity for oxygen Heme is located in the hydrophobic site. when it is in the lungs, and where the pressure is around 100 torr. When it reaches Heme Fe interacts with His. the tissues, where it's around 20 torr, the Coordination bond affinity for oxygen is still quite high. This makes myoglobin less efficient of an oxygen Propionates of heme interact with Arg. transporter than hemoglobin, which loses it's Ionic bond affinity for oxygen as the pressure goes down and releases the oxygen into the tissues. Heme is easily dissociated from the protein. Myoglobin's strong affinity for oxygen means that it keeps the oxygen binded to itself

His (E7) is the oxygen binding site. instead of releasing it into the tissues. 4 Sickle cell anemia & malaria resistance

In sickle cell hemoglobin (HbS) glutamic acid in position 6 (in beta chain) is mutated to valine. This change allows the deoxygenated form of the hemoglobin to stick to itself. 5 Figure 1. HO-1 Protects against Severe Malaria Using a mouse model for cerebral malaria, Ferreira et al. (2011) suggest a biochemical basis for the link between sickle cell disease and severe malaria. The figure shows a molecular pathway that may explain why carriers of the sickle cell trait, who are heterozygous for the mutation that causes the disease (HbS), may have more resistance to severe malaria symptoms. Mice harboring the human HbS allele have elevated levels of free heme in the blood. Free heme is toxic and can cause oxidative damage, but its effects are suppressed by the upregulation of heme oxygenase-1 (HO-1), which converts heme into the antioxidant molecules carbon monoxide (CO) and biliverdin and releases iron to bind to ferritin H chain (FtH). HO-1 expression is regulated by Nrf2. In individuals with the sickle cell trait, the elevated levels of carbon monoxide prior to infection may inhibit pathogenic CD8+ T cell immune responses and also prevent Cell 145, 335 (2011). oxidative tissue damage during severe malaria. 6 - - External axial ligands: O2, CO, NO, CN , H2O, OH Distal side

O2 O2 NO OH-

Fe(III) Fe(II) Fe(II) Fe(II) Fe(II)

His Cys His His Internal axial ligands from protein: His, Cys Proximal side

6-coordinated 5-coordinated

Ligand field - - Strong: O2, CO, NO – heme Fe(II), OH , CN - heme Fe(III) Low spin complex

- 3- Weak: vacant, H2O – heme Fe(II), H2O, F , N - heme Fe(III) High spin complex 7 Iron Protoporphyrin IX, heme b Heme a Prosthetic group Heme a is a form of heme found A flat and planar structure. In cytochromes a and a3. -. Itself Toxic O2 , H2O2 are formed. Insoluble 8 6-coordinate low-spin

Cytochrome c has two axial ligands to the heme iron complex.

Cytochrome c does not have the vacant pocket to receive O2 or CO. 9 Cytochrome c has no O (CO, NO) binding pocket, different from myoglobin and hemoglobin. 2 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 10 reactive oxygen species in triggering the cell-death pathway and describe an early role for cytochrome c before caspase activation. - - External axial ligands: O2, CO, NO, CN , H2O, OH Distal side

O2 Met NO OH-

Fe(III) Fe(II) Fe(II) Fe(II) Fe(II)

His His His His Internal axial ligands from protei: His, Cys Proximal side

6-coordinated 5-coordinated

Ligand field - - Strong: O2, CO, NO – heme Fe(II), OH , CN - heme Fe(III) Low spin complex

- 3- Weak: vacant, H2O – heme Fe(II), H2O, F , N - heme Fe(III) High spin complex 11 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 (not complex III! Upper is wrong!) the electrons are passed to oxygen (O2) to make water. This final step is why you need oxygen to live.

12 Complex III 13 Ubiquinone and the Proton Pump

Ubiquinone is a lipid soluble cofactor that accepts and donates electrons in oxidation- reduction 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.

14 Complex III

15 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.

16 Cytochrome c oxidase or Complex IV is a large transmembrane protein complex Found in bacteria and mitochondria.

2+ + 4 Fe -cytochrome c + 8 H in + O2



3+ 2 + 4 Fe -cytochrome c + 2 H O + 4 H out

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

18 Complex IV

Two electrons are passed from two cytochrome c's, through the CuA and cytochrome a sites to the cytochrome a3- CuB 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 O2. The oxygen is rapidly reduced, with two electrons +2 coming from the Fe cytochrome a3, which is converted to the ferryl +4 oxo form (Fe =O). The oxygen atom close to CuB 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 ...... 19

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.

20 Cytochrome c has no O (CO, NO) binding pocket, different from myoglobin and hemoglobin. 2 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 21 reactive oxygen species in triggering the cell-death pathway and describe an early role for cytochrome c before caspase activation. Apoptosis – the programmed death of a cell

22 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 anti- apoptotic molecules Bcl-2 and Bcl-XL 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 death- domain protein; FASL, FAS ligand; FLIP, FADD-like IL-1- converting enzyme-inhibitory protein; MCL1, myeloid-cell leukaemia sequence 1; SMAC, second mitochondria- derived activator of caspase.

23 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.

24 Nature Struc. Biol. 10, 983 (2003).

Figure 1. A representative signaling cascade of the mitochondria- mediated apoptosis. X-ray irradiation causes double- strand 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 IAP- mediated inhibition of caspases. In this diagram, the signaling steps prior to and after mitochondria are colored blue and orange, respectively. Although not shown, the anti- apoptotic Bcl-2 and Bcl-xL also reside in the outer membrane of mitochondria.

25 PNAS, 102, 17545 (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, dADP- bound Apaf-1 forms the inactive aggregate.

26 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. 27

1. Interaction with cadiolipin dissociates the axial ligand of cytochrome c.

2. Oxygen binding site is generated by cadiolipin.

-. 3. Cytochrome c interacts with H2O2 (or generated O2 and/or H2O2).

28 Peroxidase

ROOR’ + electron donor (2 e-) + 2H+  ROH + R’OH

Cytochrome c peroxidase (CCP)

+ CCP + H2O2 + 2 ferrocytochrome c + 2H  CCP + 2H2O + 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

29 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 (O2) or hypoxia have been studied extensively, the precise identity of mammalian cellular O2 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 O2 sensing and subsequent HIF-1α and HIF-2α stabilization at 1.5% O2. In the absence of this signal, HIF-α subunits continue to be degraded. Furthermore, exogenous treatment with H2O2 or severe O2 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 O2 sensing pathway.

30 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 31

.....characterization of the active site structure of oxidatively modified forms of cytochrome c (Cyt-c) free in solution and in complexes with cardiolipin (CL). The studied post-translational modifications of Cyt-c include methionine sulfoxidation and tyrosine nitration, which lead to altered heme axial ligation and increased peroxidase activity ...... binding to CL liposomes induces in all cases the formation of a spectroscopically identical bis-His axial coordination conformer that more efficiently promotes lipid peroxidation...... thus suggesting a labile distal His ligand as the basis for the CL-induced increase in enzymatic activity observed for all protein variants. For Cyt-c nitrated at Tyr74 and sulfoxidized at Met80, the binding affinities for CL are ∼4 times larger than for wild-type Cyt-c. ...we propose that these post-translational modifications may amplify the pro-apoptotic signal of Cyt-c under oxidative stress conditions at CL concentrations lower than for the unmodified protein.

Biochemistry 54, 7491 (2015) 32 Alternative Conformations of Cytochrome c: Structure, Function, and Detection Biochemistry 55, 407 (2016)

Figure 5. Phospholipid binding sites in horse cyt c. (a) Cartoon representation of horse cyt c (PDB 1HRC) depicting phospholipid binding sites A, C and L. Site A (magenta) comprises residues Lys72, Lys73, Lys86 and Lys87, site C (cyan) is made up of key amino acid residue Asn52, and site L (yellow) incorporates amino acid residues Lys22, Lys25, Lys27, His26 and His33. Sites A and L interact with lipid structures mainly via electrostatic interactions whereas site C does so through hydrogen bonding contacts. (b) Surface map representation of horse cyt c illustrating solvent accessibility and geometry of the phospholipid binding sites. 34

Figure 7. Alternative conformations of cyt c bound to cardiolipin. The reversible association of cyt c with CL has been probed via single-labeling techniques. Fluorescent dyes placed in each of four highly flexible folding units (positions 4, 39, 66 and 92) reported on conformational changes that occur upon binding to CL. In the presence of CL, cyt c transitions from the native state into two alternative conformers that differ in their degree of compactness. The cyt c-CL complex is sensitive to high ionic strength and oxidation of CL, leading to dissociation from the lipid structure. From Hanske et al, Proc. Natl. Acad. Sci. 2011, with permission.

36 37 38 Molecular Basis and Cosequences of the Cytochrome c-tRNA Interaction

J. Biol. Chem. in press. Published on March 9, 2016 as Manuscript M115.697789

The intrinsic apoptosis pathway occurs through the release of mitochondrial cytochrome c to the cytosol, where it promotes activation of the caspase family of proteases. The observation that tRNA binds to cytochrome c revealed a previously unexpected mode of apoptotic regulation. However, the molecular characteristics of this interaction, and its impact on each interaction partner, are not well understood. Using a novel fluorescence assay, we show here that cytochrome c binds to tRNA with an affinity comparable to other tRNA protein binding interactions and with a molecular ratio of ~3:1. Cytochrome c recognizes the tertiary structural features of tRNA, particularly in the core region. This binding is independent of the charging state of tRNA, but is regulated by the redox state of cytochrome c. Compared to reduced cytochrome c, oxidized cytochrome c binds to tRNA with a weaker affinity, which correlates with its stronger pro-apoptotic activity. tRNA binding both facilitates cytochrome c reduction and inhibits the peroxidase activity of cytochrome c, which is involved in its release from mitochondria. Together, these findings provide new insights into the cytochrome ctRNA interaction and apoptotic regulation.

39 Figure 1

r A B o

5’-Cy3 . 5’-Cy5

Cys Val Met Phe M

E. Coli tRNA E. Coli tRNA Human tRNA Human tRNA a

1 C 6 3’-2AP D-loop -Prf

O O O OOH OH D H H H HH Prf H 5'-tRNA 5'-tRNA HN HN 5'-tRNAHN 5'-tRNA HN H H 5'-tRNA NH2 5'-t5R'-tNRANA NH2 5'-tRNA H O NAD+/NADP+ O NAD+/NADP+ H O O O O O O H H N NADH/NADPH N NNADH/NADPH O N OO O H O H O R H H H H N H N H R O OH H O NaBH HOH NaBH 4 H H Profl4avine H H Proflavine H H FAD, Dus1p FAD, Dus1p O HOOH O Step 2 StepS t2ep 3 Step 3 H H Step 1 H H H H Step 1 H H O OH O OH H HH H H H O OH O OH H H 3'-tRNA 3'-tRNA O OH OOOHOH O OH 3'-tRNA 3'-tRNA 3'-tRNA 3'-t3R'-tNRANA 3'-tRNA

A B D Figure 4

0.9 D

100 o

-1 w n + Cyt c (14 s ) Cys n

l o

tRNA + cyt c o i 5 a

d s 0.8

e r

95 (K = 1.0 ± 0.3 mM)

d d

) e

f 4

r v

o 0

n 0.7 4 1

Cys o

tRNA + CCA + cyt c h x

c -1) 90

t (

- Cyt c (15 s

(K = 0.9 ± 0.3 M) /

m )

/ e c d 0.6 w

c 3 u

M n

w d 85 µ

( o

r c

c 0.5 d

p s

2 o

f e

g r

/ o

o 80

n u

0.4

g l

o 1

i F

e t

t c

75 o a

0.3 n r

0 M F

c 0.2 h 70 Charging - +

-0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 0 5 10 15 ,

Time (s) Cytochrome c (mM) 1

C E CCA-adding enzyme 100 tRNACys + cyt c

(Kd = 1.0 ± 0.3 mM)

3’-CCA end 95

) 4

0 Cys

1 tRNA + CysRS + cyt c

x 90 (

(Kd = 1.2 ± 0.3 mM)

e c

n 85

e c

Cyt c s

e r

o 80

l F CysRS 75

anticodon 70 0 5 10 15 Cytochrome c (mM) 41 Cytochrome b5

42 Cytochromes b5 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 b5 reductase + + NADH + H + 2 ferricytochrome b5 = NAD + 2 ferrocytochrome b5 L-ascorbate + ferricytochrome b5 = monodehydroascorbate + ferrocytochrome b5

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 O2. Fe(III) does not bind to O2.

43 Rat cytochrome b5

Human cytochrome b5

44 Cytochrome P450 45 46 Cytochrome Cytochrome b5 b5 reductase

47 Fig. 1. The elements of redox chains which are entered into the two- membrane 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) 48