University:
Amherst College
Course:
CHEM 330 | Biochemical Principles of Life at the Molecular Level
Academic year:

2020
Views:

499

Pages:

16
Author:

Amina Clayton

BioChem 330 - Course Outline • Metabolism and Bioenergetics (II) – ENZYME CATALYSIS: • kinetic constants kcat, Km • Catalytic strategies, the serine proteases – CATABOLISM (breakdown) • Carbohydrates – – – – Glycolysis Tricarboxylic Acid Cycle Electron Transport Chemiosmosis and ATPase • Fatty acids and amino acids Overview for Today, November 15, 2011 •Electron Transport Players •Diagram of the Chain •Complex 1…. •NADH oxidase/ubiquinone reductase •Complex III …… • ubiquinol oxidase/cyc c reductase A reminder, things above reduce things below... Note sign change • So: – NADH NAD+ + H+ + 2e– UbiQ + 2H+ + 2e-  UbiQH2 – NADH + UbiQ + H+  NAD+ UbiQH2 • Eo’ = + 0.32 V Eo’ = +0.045 V ΔEo’ = + 0.365 V And ΔGo’ = - nFΔEo’ = - (2)(23.1 kcal/V*mol)(.365) = -17 kcal/mole An overview of what happens to NADH... Complex IV Large molecules Complex III • Small molecules Complex I Electrons are transferred from NADH to O2 through a chain of three large protein complexes called NADHQ reductase, cytochrome reductase, and cytochrome oxidase. Electron flow within these transmembrane complexes leads to the pumping of protons across the inner mitochondrial membrane [and thereby to the synthesis of ATP]. Ubiquinone Note that two protons need to be added to convert Q to QH2 • • • Small molecule Hydrophobic, soluble in the mitochondrial membrane Carrier of both electrons/ 2H+ …+ 7 more isoprenoid units Cytochromes… (carry only 1 electron) Differences in the protein environment of the heme group in various cytochromes make it more or less costly to remove an electron: [Fe2+  Fe3+ + 1 e-] • Differences in that energy cost correspond to differences in Eo’. • Cytochrome C is shown at left. How we know what we do about the chain... • Eo’s can be determined, and ordered from stronger reducers to stronger oxidizers in situ using inhibitors • Oxi/red can be followed optically from both Soret band at about 410 nm and the longer λ bands which are actually a bit more sensitive. At long λ, oxidized cyt c (Fe+3) has only one band (530 nm) but reduced (Fe+2) has an alpha (550 nm)and a beta (510 nm). • FeS clusters can be monitored by their EPR signal, which comes from the unpaired electrons – Fe(III) (d5, gives either five or one unpaired electron depending on splitting) – Fe(II) (d6, either 4/2 or zero unpaired edepending on splitting) The electron transport chain • Looks energetically like this… … and functionally like this (although not necessarily clustered nicely like so…) Complex I,II: Passing electrons to ubiquinone • • Complex I • – Electron carriers FMN, 4Fe-4S center, bound UbiQ, 2Fe-2S center – over 30 protein chains Complex II EM reconstruction IN OUT 2 electrons from NADH (through Complex I) …or 2 from succinate to FADH2 (through Complex II, see TCA 5) Complex I • • • Structure unknown, except by EM Donates 2 electrons to ubiquinone Translocates 2 protons from in to out Complex I…..What do we know about accepting electrons from NADH and donating them to ubiquinone? • most intricate membrane-bound enzyme known to date, 40 years since the first isolation from bovine heart mitochondria – 43 different subunits whose primary structures have been determined (7 are encoded by mitochondrial DNA) – one noncovalently-bound FMN molecule – 2-3 binuclear (Fe2S2) and 6 tetranuclear (Fe4S4) iron-sulfur clusters – three unique quinone sites are the basis of the reaction to inhibitors Complex III: Collecting the electrons from QH2 - The cyt b/c1 complex • Cytochrome b/c1 complex - the electron carriers – Carrier #e - Eo’ (V) – (UbiQ) 2 0.045 e donor – cyt b 1 0.077 – Fe-S ? ? – cyt c1 1 0.22 – (Cyt c) 1 0.25 e acceptor • Which suggests a sequence of transfers • Q  cyt b  Fe-S  cyt c1  cyt c Complex III: Collecting the electrons from QH2 - The cyt bc1 complex Complex III: Collecting the electrons from QH2 - The cyt b/c1 complex • Q  cyt b  Fe-S  cyt c1  cyt c • Above scheme is also consistent with these results e donor – In the presence of antimycin, an inhibitor of etransport,, reduced QH2 and cyt b accumulate, so antimycin should block between cyt b and Fe-S Protein/Cyt c1 – Removal of Fe-S-Protein also results in accumulation of reduced cyt b and oxidized cyt c1 e acceptor Complex III: Cytochrome b/c1 • • The b/c1-complex from beef heart mitochondria Subunit Redox centers Mr(beef) Function • • • • • • • • • • • • • • Core I Core II Cyt b (III) none none heme bH heme bL 53.6 46.5 42.6 Cyt c1(IV) Rieske (V) heme c1 2Fe.2S center 27.3 21.6 Subunit VI Subunit VII Subunit VIII Subunit IX Subunit X Subunit XI none none none none none none 13.3 9.5 9.2 8.0 7.2 6.4 No catalytic, protein transport No catalytic, protein transport donor to Qi-site acceptor from SQ at Qo-site transmembrane elec transfer donor to cyt c acceptor from QoH2 donor to cyt c1 none known none known hinge protein (interacts w c1) none known none known none known Complex III: Cytochrome b/c1 How does it work? One subunit minus SU1 and SU2  Matrix side N Cytoplasm side P  Please note this image is inverted from previous bc1 image QH2 + 2 ferricyt c3+ + 2H+N <==> Q + 2 ferrocyt c2+ + 4H+P  Net reaction is to remove two electrons from ubiquinol to make ubiquinone in the membrane and to give 2 electrons to two molecules of cyt c (Fe(III) → Fe (II)) in the cytoplasmic/ intermembrane space (or periplasm in bacteria) [NOTE THAT THIS DIRECT TRANSFER IS NOT WHAT HAPPENS] (vector) 2 H+ are consumed from the matrix (N side) and end up across the membrane in the cytoplasmic (P side) (scalar) 2 H+ are released into the inter membrane space from every QH2/Q oxidized for a net of 4H+ per cycle Nothing in life is simple.. • • • • Q  cyt b (anti) Fe-S  cyt c1  cyt c when antimycin is added, reduced cyt b builds up, cyt c not reduced when Fe-S is removed, reduced cyt b builds up, cyt c not reduced but when Fe-S if removed and THEN antimycin is added to the Fe-S deficient complex, reduction of cyt b is blocked??????? • Not only that, there are, in fact, two hemes in cyt b, one (bL) with a lower electron affinity than the other (bH) • A proposed solution - the Q cycle: Antimycin blocks here And then..

BioChem 330 - Course Outline Electron Transport Part 1

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