CYTOCHROMES

1. CYTOCHROMES M.Prasad Naidu MSc Medical Biochemistry, Ph.D.Research Scholar

2. Cytochromes Cytochromesare electron carriers containing hemes . Hemes in the 3 classes of cytochrome (a, b, c) differ in substituents on the porphyrin ring. Some cytochromes(b,c1,a,a3) are part of large integral membrane protein complexes. Cytochrome c is a small, water-soluble protein.

4. Heme is a prosthetic group of cytochromes. • Heme contains an iron atom in a porphyrin ring system. • The heme iron can undergo 1 e- transition between ferric and ferrous states: Fe3+ + e- Fe2+ • Copper ions besides two heme A groups (a and a3) act as electron carriers in Cyta,a3 Cu2++e- Cu+

5. Cytochromes proteins that accept electrons from QH2 or FeS Ultimately transfers the electrons to oxygen

6. Mitochondrial Complexes NADH Dehydrogenase Succinate dehydrogenase Cytochrome Oxidase CoQ-cyt c Reductase

7. Mitochondrial respiratory chain: • Complex I: • - Transfers e- from NADH to quinone pool & pumps H+. • Complex II: • - Transfers e- from succinate to quinone pool. • Complex III: • - Transfers e- from quinol to cytochrome c & pumps H+. • Complex IV: • - Accepts e- from cytochrome c, reduces O2 to H2O & pumps H+. • Complex V: • - Harvests H+ gradient & regenerates ATP.

8. Complex III (or bc1-complex) • Catalyses the transfer of e- to cytochrome c. • Pumps protons through redox coupled Q-cycle. • - Coupled ubiquinone/ubiquinol redox reactions occur either side of the membrane. • - Quinone/quinol binding sites labeled QP and QN (P=positive; N=negative). • Selectively diverts e- from QP to either cytochrome c or to QN

9. Q-cycle • Quinol binds at QP and e- transfer to heme c1. • Second e- transfer to bound quinone at QN via hemes bL and bH. • Quinone replaced by quinol at QP. • Above steps repeated. • Heme c1 fully reduced (passes 2e- to cyt. c). • One quinol consumed (ie. two consumed but one regenerated). • 4 H+ released to cytoplasm and 2 H+ taken up from matrix.

11. Complex IV (or cytochrome c oxidase) • Catalyses the transfer of e- from cytochrome c to O2. • Energy liberated pumps protons through conformational changes. • - Reduction of oxygen to water one of the most important reactions in biology. • More H+s taken up from matrix side, which balances bc1-complex, for which more H+ released to cytoplasmic side.

12. Cytochrome c oxidase Cu(II)  Cu(I) e- from cyt c to a Heme A and Cu act together to transfer electrons to oxygen

13. Distribution of cofactors • Subunit I: heme a and the binuclear centre (heme a3 & CuB). • - It is the binuclear centre which forms the active site for O2 reduction. • Subunit II: dinuclear centre (CuA which is two Cu atoms). • - Electrons first transferred from cytochrome c to CuA. • - Passed onto the binuclear centre via heme a. • Also a Mg ion present at the interface between subunits.

14. NOsynthase CYP11A1,B CYP17 CYP19 CYP21A2 CYP7B1 CYP26A1,B1,C1 CYP27A1, B1, C1 CYP24 Retinoic acid CYP51 CYP7A1 CYP27A1 CYP46 CYP39 Steroid(hormone)s Vitamin D CYP5A1 CYP8 A1,B1 Endogenous Substrates Arachidonic acid Sterols CYP CYP4 A(11,22),B1,F(2,3,8,12,22), V2,X1,Z1 Xenobiotics CYP1A1, A2, B1 CYP3A (4,5,7, 43) CYP2 A(6,7,13),B6,C(8,9,18,19)D6,E1,F1,J2,R1,S1,U1,W1

15. Ethanol Metabolism • Ethanol is oxidized to acetaldehyde through several enzymatic pathways: • Alcohol dehydrogenase • CH3CH2OH + NAD+→ CH3CHO + NADH + H+ • Catalase • CH3CH2OH + H2O2→ CH3CHO + 2 H2O • Cytochrome P-450 • The “Microsomal Ethanol Oxidizing System” (MEOS) • CH3CH2OH + NADPH + H+ + O2 → CH3CHO + 2 H2O + NADP+ • And through a non-enzymatic free radical pathway:

16. Apoptotic Pathways Effectors and Modulators • There are two major apoptotic pathways in mammalian cells. • The death receptor pathway, exemplified by FasL binding to an extracellular receptor, causes the formation of the DISC that results in the activation of caspase-8. • The mitochondrial pathway is activated by most cellular stresses. A resulting signal or intracellular change causes the release of cytochrome c into the cytosol. Cytochrome c binds to Apaf-1 and procaspase-9 to form the apoptosome and catalyzes the activation of caspase-9.

17. Mitochondrial Pathway Death Receptor Pathway ceramide others oxidants FasL Fas/Apo1 /CD95 DNA damage D D Bcl-2 D D D FADD DISC Procaspase 8 Apaf -1 BID Procaspase 9 Cytochrome c Caspase 8 Procaspase 3 Apaf -1 dATP dATP Caspase 3 Cellular targets apoptosome Caspase 9 Major Apoptotic Pathways in Mammalian Cells

18. Initiator caspases, such as 8 and 9, activate effector caspases that cleave multiple cellular proteins. Caspases are characterized by an active site cysteine. • Bcl-2 is a proto-oncogene that was first discovered in B-cell lymphoma. Bcl-2 prevents apoptosis by blocking the release of cytochrome c from the mitochondrion by an unknown mechanism. There are many Bcl-2 homologs, some with pro- and others with anti-apoptotic functions. The ratio between these two types helps determine the fate of the cell. Additional information about Bcl-2 family members can be found

19. Cytochrome c and Cellular Redox Environment • Cytochrome c in solution can act as an antioxidant and an ROS scavenging function for cytochrome c in the intermembrane space has been proposed by Skulachev. • Release of cytochrome c into the cytosol from the mitochondrion interrupts the electron transport chain resulting in increased production of superoxide from the mitochondrion.

20. Binding of cytochrome c to form the apoptosome and activate caspase-9 does not appear to depend on the ability of cytochrome c to transfer or accept electrons. • However, the reduction state of cytochrome c may still be important because reduction and oxidation cause conformational changes that may be critical for cytochrome c binding to Apaf-1 and procaspase-9.

25. THAN’Q