MEMBRANE TRANSPORT & BIOENERGETICS Richard Neutze Richard.Neutze@chembio.chalmers.se Ph. 773 3974 http://www.csb.gu.se/neutze/bioenergetics.html Aims Present a detailed analysis of the mechanisms of energy transduction within the cell. Emphasis placed on the interplay between structure & function for membrane proteins governing energy transduction. Tutorials: Aim to provide skills & resources for the literature project. • Using structural & citation data bases. • Packages for structural analysis. • Making figures. • Writing scientific reports. • Presentation of literature study. Literature study will: • Work in pairs. • Choose one structure-related research article. • Ask what new information concerning the functional mechanism of this protein has emerged from its structure? • Write a report of not more than eight pages. • Give a 15 minute presentation of the project. Course Assessment: 50 % written exam. • Wednesday December 12th - time we discuss now. A literature project. • 40 % written project. - Due Monday December 6th. • 10 % presentation. • Thursday December 2nd. Grades 5, 4, 3 or fail. Science: the way it is! •1900 Physicists thought they were close to a theory of everything. • Newton gravitation worked well. • Electricity & magnetism unified by Maxwell. • Milky Way seemed to encompass everything. • Within the first two decades of 20th century: • Quantum Mechanics. • Theory of Relativity. • Other galaxies discovered. • 21st century: the century for biology! • Piecing together the jig-saw of life will uncover numerous remarkable phenomenon. The Big Bang •14 billion years ago the universe began from a point. - Quantum fluctuations ``borrowed'' just enough from the vacuum to kick it all off. - Positive mass energy + negative gravitational = 0! • After ~300 thousand years hydrogen condensed from a plasma. - Stars condensed from hydrogen. -Inhomogeneities led to galaxy formation. • 200 billion stars in a galaxy. • 100 billion galaxies in the universe. Star cycle: • Stars ``burn'' hydrogen: • Hydrogen fusion product is helium. • Fusion products of helium & hydrogen etc. = heavier atoms (eg. carbon, oxygen, nitrogen etc). • When stars ``burn out'' they collapse. • Massive gravitationally driven heating. • Heating drives supernova (huge explosions). • More complex atoms created. Solar system creation: • Began after one (or more) local supernova 4.6 billion years ago. - Our sun a second or third generation star. • Inner planets formed from collisions of moon-sized planetismals. • Venus, Earth & Mars received similar inventories of C, O & H2O. • Mars still has frozen water below the surface. • Thin (not massive enough) CO2 atmosphere & cold! • Venus lost water due to a runaway greenhouse effect. - Water vapor in the high atmosphere is photolysed into H2 & O, & H2 lost to space. • Thick CO2 atmosphere & hot (500oC)! Earth: • Most CO2 in carbonate materials (eg. limestone). - CO2 blanket much less & no runaway greenhouse effect. - Water on the surface. • Volcanic activity. - Recycles the biosphere. • Magnetic field. - Protects against solar (ions) radiation. Moon: • Pro-Earth struck by an inner planet ~Mars 4.5 billion years ago. - Ejected molten mantel into the orbit. - Some coalesced into the moon. • Gave the earth its spin & tilt. - Heating & cooling pattern (day/night & seasons) critical. • Exaggerated tides. - Powers plate tectonics & volcanism. - Volcanism provides reducing cations & recycles biosphere. • Role in evolution? Early bioenergetics: • First life about 3.8 billion years ago. • Earliest replicating system possibly an RNA molecule. - RNA world located in rock pores around volcanic springs. - Lived on redox contrast of more oxidised atmosphere/ocean & more reduced fluids in contact with volcanic magma. - Dissolved suplhate provide oxidation power for organisms to react against hydrothermal fluids, eg. hydrogen & methane. • ATP addiction probably emerged from the RNA world. - Ribozymes (catalytic RNA) can polymerise RNA-NTP molecules (NTP = nucleotide tri-phosphates). - 14 nucleotide addition with 97 % fidelity was demonstrated. - Early metabolism of RNA world based on an NTP. - Triphosphate bond releases 10 kcal/mol & is stable (10 -10/min). Cell membranes: • RNA based life contained some cellular compartmentalization. - Compartments keep replicase & genomic RNA together. - Evolutionary advantages remain with the organism which produced it. • Cells in contemporary biology surrounded by amphiphatic lipids. • In early life compartmentalization could be achieved by: - Organising centres (like a rybozome). - Organisation along a surface. - Passive compartmentalization within pores of rocks, or surface of fine particles. DNA & proteins: • RNA world invented of protein synthesis. - Crowning achievement. - Protein synthesis instructed & catalysed by RNA. - Rybosome has RNA @ amino-acid polymerisation active site. - Proteins more versatile & efficient in catalysis. • DNA more chemically stable than RNA. - Allows larger genomes. • DNA not catalytically active. - Forms a partnership with proteins. Evolution of photosynthesis: • Accidental use of pigments where disequillibria easily exploited. - Anoxygenic photosynthesis may have evolved from bacterium using infrared thermotaxis. - Organism drifted into shallow water & used sunlight. - Later evolution of an oxygen generating complex exploiting Mn4O4 to Mn4O6 chemistry to generate O2 from H2O. • 3.5 billion years ago cyanobacteria evolved. - Probably a genetic exchange between interdependent green & purple bacteria. - Created an organism which could live freely on the planet wherever H2O, CO2 & light available. • Tremendous increase in the biosphere. Rubisco & carbon dating: • Rubisco is an ancient enzyme which fixes atmospheric CO2. - When there is excess it prefers 12C to 13C. - Measuring 12C to 13C ratio in rocks the standard method of carbon dating. • Rubisco predates oxygenic photosynthesis. • A qwerty enzyme. - The qwerty keyboard - designed to avoid mechanical jamming & far from optimal. - Rubisco fixes CO2, but may also fix O2 (ie. undo the benefits of photosynthesis). - This ineffeciency maintains reasonable CO2 levels in the atmosphere. Change of atmosphere: • Debate as to when the atmosphere became O2 rich. - Entire biosphere now ``rich'' in redox potential. • Oxygenic photosynthesis appeared 3.5 billion years ago. - Global oxygen production similar order of magnitude ever since. - But levels depend on consumption also! • Evidence that O2 levels increased 2.2 billion years ago. - Possibly due to complex eukaryotes. • Biodiversity took off. Four aeons: • Hadean: - From formation of the planet to early life. • Archaean: - Prokaryotic life. • Proterozoic: - First eukaryotes. • Phanerozoic. • Life visible without need of a microscope. Amphipathic molecules • Solubility depends on favorable interactions with water. - Hydrogen bonds. - Ionic interactions. - Polar compounds. •Hydrocarbons are non-polar, non ionic & cannot form H-bonds. - Hydrophobic not hydrophilic substances. • Amphipathic molecules have a hydrophilic head group & a hydrophobic tail. - Form mono-layers, micelles and bilayer vesicles. Lipid bilayers • Lipids have polar hydrophilic ``head'' & hydrophobic ``tail''. • Form lipid-bilayer membranes. - Most biological membrane lipids have two hydrocarbon tails. - Divide different compartments of the cell. - Allow non-polar molecules to move within the membrane. - Prevent the diffusion of polar molecules across the membrane. - Can with-stand 200 mV over ~ 40Å (50 MV/m). • Glycerophospholipids. - Major class of naturally occurring phospholipids (phosphate containing head-group). Transport across membranes • A cell is neither entirely open or closed to its surroundings. - Interior of cell protected from toxic substances. - Metabolites imported into the cell. - Waste products extracted from the cell. - Changes in cell environment detected. - Inter-cellular signals transmitted. • Passive transport. - Accomplished by the random diffusion of molecules through the membrane (non-polar molecules only). • Facilitated transport: - Ion pores, which define ``holes'' in the membrane. - Carrier molecules, which form a hydrophobic casing. - Both allow only specific ions to move through the membrane. • Active transport. - Integral membrane proteins use chemical or light energy to pump ions against a concentration gradient (eg. H+ or K+). Modern bioenergetics • Biology harvests the energy content of light. - Photosynthesis → chemical energy. • ATP is the basic ``energy currency'' of the cell. - Produced by energy-transducing membrane proteins. - Protons are first pumped across a cell membrane. - ATP-synthase harvests the proton concentration gradient to generate ATP from ADP + Pi. Chemical energy to light Light to chemical energy Mitochondria • Found within eukaryotic cells. • Primary producer of ATP. - Basic ``workhorse'' of the cell. • Typically 0.7 to 1.0 mm long. • Outermembrane has porins allowing free access of small particles. • Approximately 500 mg/ml of the inner membrane is protein. Respiratory chain of mitochondria • Membrane proteins transfer e- from NADH or Succinate to O2. - Electrons enter at complex I or II. - Complex I, III and IV pump protons. - Complex IV reduces O2 to H2O. • Complex V (ATP-synthase) uses the proton-gradient to generate ATP from ADP & Pi. ATPsynthase • ATPsynthase harvests the H+ gradient to regenerate ATP from ADP & Pi. - Back diffusion of H+ causes membrane portion to rotate. - Mechanically coupled to the soluble portion. - Maintains the ATP/ADP ratio 10 orders of magnitude from equilibrium. - Excess ATP used as the energy currency of the cell. • Mechanism conserved throughout biology. Summary of lecture 1: • Universe 14 billion years old. • Earth/solar system 4.5 billion years old. • First life appeared 3.5 billion years ago. • ATP addiction inherited from RNA world. • Overwhelming source of ATP is highly a conserved process in energy-transducing membranes. • To understand the bioenergetics must understand proton translocation processes.