Bioenergetics Flashcards

Question

Cytochrome b6f and Photosystem I

Answer 1

Cyt b6f: f(x) similar to complex III- oxidises Q, passing e- to small soluble protein, operating a Q cycle. Also structurally similar. Cyanobacteria Q/b6f/c6-PC shared between respiratory+photosynth pathway. PSI: accept e- from plastocyanin or c-type cyt in some algae/ cyanobacteria-> P700 chlorophyll special pair->2 monomeric Chla+ phylloquinone Q. Pass 3 different Fe4S4 clusters Fx,a,b, -> ferredoxin. PSI= heterodimer of PsaA+PsaB, analogous to DI+D2 in PSII. Whole complex isolated as a trimer. Cyclic transfer round PSI also possible: doesn’t produce NADPH, allowing balancing needs for ATP+ reducing equivalents; increases lumen acidification (regulatory response?); uses e- from ferredoxin passed to PQ; 2 routes: PGR5+ PGRL1 or membrane complex (similar to complex I) w/ peripheral arm.

Answer 2

Evolution/diversity: Roles of complexes+ differences between cyanobacteria+ chloroplasts unclear. Green sulphur bacterium Chlorobium has photo machinery similar to PSI (e- out through FeS clusters-> ferredoxin); no PSII equiv, but e- suppliable back to reaction centre, e.g., by ox H2S->S. Overall, diversity of PSs can be categorised to PSI/PSII-type. In cyanobacteria, b6f complex same as for respiration (chloroplasts got it from cyanobacteria- endosymbiosis).

Answer 3

Safety valves: E abs+ not used for photochemistry dangerous, +damage possible esp to PSII even if used. Safety valves can quench unused E (non-photochemical quenching/ NPQ): * Xanthophyll cycle- high light-> favour conversion xanthophyll carotenoid violaxanthin-> zeaxanthin (reversible de-epoxidation)- enhances quenching. Violaxanthin associated w/ light harvesting complex, well placed to modulate f(x). * PsbS: antenna protein that is pH sensitive- enhances NPQ by unclear mech, likely synergistic effect w/ violaxanthin. Chloroplast antenna don’t have clear homologues in photobacteria, but similar to cyanobacterial high light inducible proteins (HLIPs)- potential part of stress response * PSI/II have diff abs (700 vs 680nm), may run @ diff rates. In chloroplast, if PSII too fast, light harvesting antenna shifts away from it+ vice versa (‘state transitions’) due to kinase responding to redox state of PQ pool (PQ pool reduction-> kinase activation-> Pi LHCII-> LHCII leaves PSII). Cyanobacteria have equivalent state transitions where phycobilisome decoupled from 1/other PS. * Phycobilisome assoc. ‘orange carotenoid protein’ may modulate its f(x)

Answer 4

Safety valves may also help discharge the ETC: * Chloroplasts have terminal oxidase (similar to mt)- passes e- from reduced PQ->O2 * Cyanobacteria respiratory ETC in same mem as PSs, incl PQ, cyt b6f, plastocyanin. Intersection-> respiratory chain can take xs e-s+ pass to O2 (esp important in fluctuating light) * Some species- flavo-diiron proteins- transfer xs e- direct/indirectly to O2 in ctrled way. Flv1/3 protect PSI (cyanos and many euk algae), Flv 2/4 PSII (some cyanos)

Answer 5

“chemiosmotic hypothesis” proposes that: e- transport is coupled to H+ transport across a membrane; the membrane is relatively impermeable to protons; return of protons through the membrane (e-chemical proton gradient) is coupled to ATP synth; there are other transporters in membrane, some powered by H+ gradient General schemes for mt+ thylakoids: mt extrude protons when given O2 pulse w/ oxidisable substrate present. Chloroplasts take up e- when light. Similar experiments w/ purified mt Complexes I,III,IV, chloroplast PSI+II, bf complex show e- transport associated w/ H+ transport. 2 models for H+ translocation 1) Redox loop models- H+ carried across mem w/e- as H on same small molecule (e.g., UQ). Alternating e-+ H+ carriers on correct sides/mem-> H+ transport directly coupled to e- transport (e.g., PSs/Q cycle) 2) Proton pump model- e-/H+ flows separate, w/ indirect coupling mechanism e.g., conf change in protein Redox loop model for Z-scheme in chloroplasts (Q cycle moves H+ by redox loop too). Proton pumping by complex I is via conf changes assoc. w/e- transfer that drove H+ extrusion electrostatically. CII doesn’t transport H+. Q-cycle accepted mech for CIII. CIV transports H+ by redox loop+ proton pump mechs. Oxygenic Photosynth- proteins liberated into thylakoid lumen by H2O oxidation, translocated across by PQ+ cy tbt complex, removed from matrix by NADPH formation.

Answer 6

Permeability of membrane to H+: Permeability increased by uncouplers. Proton translocators (lipophilic weak acids, cross mem protonated or deprot) catalyse electrophoretic proton uniport across mem. If pmf exists, proton translocator cycles catalytically. @ =ve face of mem, DNP-O- protonated-> DNP-OH+ vice versa. Overall, H+ crosses mem. Proton ionophores uncouple protein translocation from ATP synthesis. Uncouplers allow rapid e- transport w/out coupled APT synth, demonstrated to act as proton ionophores provide evidence for return of H+ being coupled to ATP synth.

Answer 7

Coupling e- transport/ATP synth via pmf shown in O2 electrode. If all ADP-> ATP, pmf builds up, stopping e-transport-> mt idling (state 4). ADP added-> O2 consumption rate increases to active state 3 (ADP->ATP)+ pmf drops- shows e-transport directly linked to ability to make ATP. When nearly all ADP used up, return-> state 4- this is respiratory ctrl (Add UCP-> e-transfer resumes). Mt state transition occurs w/ work/exercise. Max exercise-> O2 consumption x10. All isolated mt have proton leak across inner mem, rate of which increases exponentially w/ pmf size, so significant in state 4 but not state 3. Respiratory ctrl ratio= state3 rate/state 4 rate= inverse measure of mt proton leakiness. Leak due to free fatty acids (cycle across mem w/ CO group carrying H+) or UCPs (generate heat in BAT- NB diff mech used in mt in plants like Titan Arum (use alternative oxidase to ox reduced Q directly w/out proton gradient)). PO ratio= mol O2 use per mol ADP use.

Answer 8

Pmf size (formula above)- delta psi calculated from measures transmembrane distribution of permeant cation @ equilibrium, like K+ in presence of valinomycin/synthetic lipi-soluble cations (TPMP+)- rapidly cross mem+ reach equilibrium. Then mem potential calc w/ Nernst equation if [K+] on each side measured. pH difference across mem calculated from distribution/permeant weak acid (e.g. acetic)- cross mem protonated but not charged- equ distribution determined by deltapH, not membrane potential. For compartments w/ acid inside (thylakoids, lysosomes), use weak bases instead.For photosynthetic mems, measurements in electrochromic shift (change in pigment st=pectral properties in response to electical potential) an be used. Mt pmf ~210mV, thylakoid 180, bacteria 100-200. Thylakoid pH and mem potential more variable (mem pot varies depending on species+lighting).

Answer 9

ATP synthase: similar across bacteria, mt, chl. Separation by mechanical treatment/salt washing-> F0+F1. F1 soluble, will hydrolyse ATP but not synth. Has 3alpha+ 3beta (globular head, ATPase activity in beta), 1 gamma(rotating crank), 1delta (holds stator arm on top of head), 1epsilon (connects gamma to F0) subunits F0: 1a (connect stator to c ring), 2b (stator arm), 8-15c (H+ translocating rotor) subunits- alone, acts as H+ channel, blockable by DCCD w/ singl Asp or Glu at centre of membrane on subunit c (suggests residue essential to proton channel). Oligomycin more widely used inhibitor of intact ATP synthase, also blocks F0 1st Crystal structure F1 ATPase beef heart 2.8A res- gamma forms bend alpha helical structure en=xtending whole length of pore up centre of F1, asymmetrically placed in pore, partly held by hyrdrophobic residue ring where the 6 alpha/beta subunits meet @F1 top. Structure stongly supports binding change mech+ dependence on rotation of gamma:

Answer 10

Binding change mechanism: pmf provides E by proton flow via F0 to make ATP+ promote release from catalytic sites where it forms spontaneously; E-linked substrate binding, formation of tightly bound ATP+ release @ 3 separate interacting sites 120o out of phase in catalytic cycle;binding changes required driven by conf changes in F1, driven by gamma, driven by c of F0. Alpha-beta pairs remain still, catalytic sites in betas @interface w/alpha. ADP+Pi bind L state; ATP forms spontaneously in T state (binds ATP better than ADP); ATP release from O state.

Answer 11

Proton flow/c rotation through F0 causes gamma rotation up tp 270Hz, visualisable microscopically. Proton enters half-channel from p side, binding c1; proton displaces R210 (glu/asp) toward adjacent c2; R210 binds c2, displacing H+ bound to it; displaced H+ (has been held longest) leaves other half channel on N side; c ring rotates, R210 remains on c2; c2 now on p-side, ready for another H+- all events concerted. Rotational can go either way, but pmf keeps it unidirectional. ½ channels ID’d on cryo-EM.

Answer 12

Gearing/ stoichiometry: #H+s=#c subunits->360 rotation->3ATP. #c/3=#H+/ATP, e.g., 8c in bovine mt-> 2.7H+/ATP. 10 in yeast, 15 cyanos. NB pmf also used for other transmembrane channels- net #H+/ATP may be even higher. NB ATP synthase may exist as dimer in mt. Directional ctrl: ATP synth driven by pmf or ATP hydrolysis to generate pmf possible. Mammals have peptide inhibitor of hydrolysis IF1 (diff from stator), binding ATP synthase likely pH-dependently. Simialr system in cyanos. Chl use redox regulation via gamma.

Answer 13

Translocators: to get ATP in/out of mt/Chl. Use adenine nucleotide translocator (well studied, 10% mt protein)- active carrier catalysing electrophoretic exchange of ATP4- out/ADP3- in across mt inner mem (net export of -ve charge favoured by pmf)- undergoes large conf change between C+M states(can be locked in 1 state by carboxyatractyloside (C) or bongkrekic acid (M)). Binding affinity to ATP/ADP diff fo C/M states so cycling-> exchange. Yeast has 35 genes in this protein family. Many other mt carriers (pi, pyruvate, di/tricarboxylate) homologous w/ like structure. Pi translocator (symporter) imports H2PO4- w/ H+, also favoured by pmf. “ATP synthasome” suggested to be formed by ATP synthase+ translocators.

Answer 14

Genome: in humans, mt genome codes Complex I,III,IV, ATP synthase, r/tRNAs-remnant of original bacterial genome- other genes lost/relocated to nucelus. Allen argues genes retained where regulation in response to redox poise of mt needed (CORR-co-location for redox reg)- may also allow reg of exp In relation to complex assembly (CES ctrl by epistasy of synth and COCOA colocation for ctrl/assembly). Huge genome size variation (plasmodium 6kb vs Arabidopsis 367kb), w/ some variations in code. Transcription usually by viral-type RNAP. Inheritance maternal: sperm mt actively destroyed in fertilised egg by UQ-dependent mech. All mtDNAs descended from that of single/ small group (“mt Eve”) living 100-200k yrs ago. Mutation+ evolution rapid (10-17x nuclear DNA) partly due to high ROS levels from ETC-> high polymorphism even in ethnically closely related groups- one reason behind extensive use in forensics+ other areas, e.g., ID remains of last 2 children of Tsar Nicholas II/Russia/ ID skeleton under Greyfriars Car park, Leicester as Richard III/ resolve racehorse breeding arguments/trace prehistoric human migration across Europe/Americas/Oceania

Answer 15

Mt diseases incl 80% MELAS (Mt encephalopathy, lactic acidosis+ stroke-like episodes) cases having substitution in mt tRNA-Leu gene; MERRF (myoclonic epilepsy+ ragged red fibres) usually sub in tRNA-Lys gene; NARP usually sub in ATP6; LHON usually mutations in NDH genes. Mt protein import: most mt proteins encoded in nucleus, translated in cytoplasm. Several destinations- need several pathways for transport. Many proteins, particularly matrix ones, have import seq cleaved off in matrix. Others have internal recognition seqs recognised by transport machinery- mt transit seq ~20-30 aa on N terminus, though seqs of diff peptides not strongly homologous, but form amphipathic alpha helix w/ +ve charges on 1 side (recognised by import machinery). Pathway of import depends on destination: 1) Most proteins enter via TOM, then: 2) May then pass-> TIM23, then: 3) can enter matrix via preseq-translocase-assoc motor PAM+ cleavage by mt processing peptidase MPP. 4) Otherwise, could be release from TIM23, (enter via TOM, stay in inner mem), or: 5) Or bypass TIM23+ go to IMS using mt IMS assembly machinery MIA or 6) Go to outer mem w/ sorting+assembly machinery SAM complex or 7) Inner mem via TIM22 or 8) Go to outer mem directly w/ mt import machinery protein 1 (Mim1) and 9) To inner mem directly by synthesis in my+ export via OXA

Answer 16

Mt structure: membranes inner, outer, Cristal (site of ox Pi). Cristae connected to inner mem by cristal junctions (regulate cell death), outer+ inner connected by contact sites (protein transport)- both depend on complex of MICOS proteins (developed @ time of endosymbiosis. Endosymbiotic origin of cristae indicated by ancient homology of MICOS). Cristal junctions may limit continuity w/ IMS-> translocated H+s may not be able to diffuse away. Complexes likely @ diff locations in cristae- ATP synthase @ apex- its dimerization may be assoc. w/ its location

Answer 17

Mt dynamics: cristal structure, shape, density can change according to cell physiological state (incl nutritional status)- probably depends on MTORC1. In some cells, continuous mt fusion/fission. Fusion dominates->mt can form single network/reticulum. Fission dominates-> 100s small particulate mt. Fusion in mammals depends on GTPases Mfn1+2 (outer mem fusion)+ GTPase OPA1 (inner). Fission requires GTPase Drp1. Fusion likely requires mem potential ->fission/fusion cycle can have role in quality ctrl of mt- damaged mt may be unable to rejoin, get degraded. Exercise can ~2x mt in skeletal muscle. Differentiation can cause changes in mt levels in cells.

Answer 18

Mt lifecycle: De novo mt synth impossible. Need to form from existing mt by fission, mtDNA rep, incorporation of new proteins+ phospholipids (mt biogenesis). Post-mitotic tissues (heart, brain) where cells no longer dividing, no net accumulation of mt, w/ turnover every ~10-25 days. Mainly degraded by autophagy (random surrounding of areas of cytoplasm by 2xmem of autophagic vesicle, which then fuses lysosomes->degradation)

Answer 19

Anaerobic mt/ organisms w/ mt w/ partially/fully lost genomes * Mt in metazoans in O2-poor env (e.g., shellfish living in sediments, parasitic worms in animal digestive tracts): use e- transport coupled to H+ translocation in mt-> ATP. Use non-O2 terminal acceptors (nitrate, nitrite, NO) or molecules generated during metabolism (=/= pyruvate->lactate/ethanol) * Hydrogenosomes: degenerate mt producing H2, typically found in unicellular euks living in guts. represent mt in varying states of evolutionary “reduction”- have many proteins typical of mt, incl protein import components. Some have other features: Blastocystis has complex I+II, II may run backward,+ alternative oxidase. Nyctotherus has remnant mt genome incl some components/ complex I * In Giardia, no complex I/II, but remnant organelle (mitosome)- functions in Fe-S cluster biogenesis These modified mt sometimes called Mt-related organelles (MROs). Monocercomonoides (anaerobic protist) doesn’t have any mt, has new pathway for Fe-S cluster biogenesis able to function in cytosol w/out need for separate compartment.

Answer 20

All contain 120-200kb genome, representing remnant genome of ancestor represented by cyanos now. Al encode components of PSI+II, b6f, ATP synthase, protein synth, ribulose bis-phosphate carboxylase (rbcL), r/tRNAs+ others. Similar arguments for gene retention to mt. Transcribed by bacterial-type RNAP (chl encoded). Land plants also have viral-type RNAP likely derived from RNAP in plant mt. Uniparental inheritance. Import pathways well-studies, some appear to have been recruited from ancestral ‘cyanobacterial’ endosymbiont. Chl structure: stroma+ thylakoids, some stacked (granal lamellae- unstacked are stromal). PSI in stromal+ ends of granal stacks; PSII in stacks, Cyt b6f equally distributed, ATP synthase stromal. Division by binary fission, many proteins ID’d- depends on mix of bacterial-type proteins+ host proteins superimposed. Evolutionary history: many photo organisms have chl w/ >2 mem- 2ndary chl where chl-containing euk was engulfed again by another euk- these chl can be traced back to endosymbiosis that gave rise to red+ green algae+ glaucophytes (which have peptidoglycan wall). Nuclear encoded proteins entering thylakoid lumen of some algae may cross up to 5 mems. Paulinella chromatophora= protist that independently invented chloroplast (its symbiont shown by phylogenetic analysis to be of cyano origin but only distantly related to green alga symbiont). Over 70 genes relocated-> nucleus, products reimported- opportunity to study independent solution to same import problem, where proteins <90aa enter w/out targeting seq+ larger proteins have this seq. Another independent chl invention claimed in marine alga- ‘nitroplast’ N-fixing organelle.

Answer 21

Non-photosynthetic chloroplasts- * developmental: proplastids, amyloplasts- convertible to chl * evolutionary: in non-photo plants (Epifagus, Monotropa)- retain chl genome. Apicomplexa (incl Toxoplasma+ plasmodium) have photosynthetic ancestry, have remnant chl w/ 35kb genome encoding ClpC+ SufB+ prteins for their exp- reason for retention controversial. Essential drug target (doxyclycline, fosmidomycin (DXR (enzyme in non-mevalonate pathway of isoprenoid biosynthesis absent in humans but present in plants, euk alae, cyanos) inhibitor) * Some species have chl w/ lost genomes (rafflesia)+ some plants/algae lost chl altogether.

Bioenergetics Flashcards

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