Microbes as Energy Transducers The Metabolic Menu Metabolic

Microbes as Energy Transducers The Metabolic Menu Metabolic Strategies Respiration & Fermentation Chemolithotrophy Photoautotrophy Biogeochemical Cycles Metabolism in Primitive Organisms

The tendency of a molecule (B) to accept an electron from another molecule is given by its electrode potential (E), a. k. a the reduction potential. E 0 is the electrode potential under standard conditions. Eh is the actual electrode potential. E 0’ is the electrode potential at p. H 7 (under biological conditions). Molecules with more negative Eh or E 0’ values are better electron donors. Electrons flow spontaneously to molecules with more positive Eh or E 0’ values.

The total free energy change occurring in these two half reactions can be calculated by the following equation: DGo’ = (-n. F)(DEh) where Eh is the free energy change under standard biological conditions, n is the number of electrons transferred, and F is the faraday constant (-96. 48 k. J/V) DEh = Eo’(oxidized partner) - Eo’(reduced partner)

Oxidation and Reduction are Coupled Reactions Negative E 0’ A becomes oxidized, losing electrons Positive E 0’ B becomes reduced, gaining electrons

Redox Rxns: Eo’= -0. 42 V Eo’= +0. 82 V

DGo’ for the formation of water: Eo’of electron donor once oxidized DEh = (+0. 82) – (-0. 42) Eo’of electron donor when reduced DGo’ = (-2)(96. 48)(1. 24) = -239. 27 k. J

Other final electron acceptor O 2 as final electron acceptor

Metabolic Menu For Chemotrophs a. k. a chemolithotrophs, chemoautotrophs a. k. a chemoorganotrophs, chemoheterotrophs

Electrons in redox reactions are carried from the primary electron donor to the terminal electron donor. Electrons are not freely diffusible in the cell. They are carried by electron carriers.

Electron carriers 1. Freely diffusible (coenzymes/cofactors) – move electrons from one enzyme to another to take part in reactions (like batteries or electron taxis) -NAD/NADH: catabolic, energygenerating reactions, transfers hydride ion (2 e- + 1 H+) -NADP/NADPH: anabolic, biosynthetic reactions 2. Fixed carriers, where carrier is a prosthetic group firmly attached to enzyme in the cytoplasmic membrane (electron transport chain)… like a power line or stops on the city bus line

Then, need to store energy…

Energy currencies for cells are usually compounds whose DG 0’ is greater than 30 k. J/mol. Anhydride bonds

Energy contained in glucose used to convert low-energy phosphoryl groups to highenergy bonds: 1, 3 bisphoglycerate Phosphoenolpyruvate Acetyl-Co. A Succinyl-Co. A 2 stages: 1. Splitting glucose (C 6) into two phosphoglyceraldehydes (C 3) 2. Oxidizing phosphoglyceraldehyde to pyruvate

Two Ways to Make ATP: Quick & Dirty Fermentation or Turbo-Charged Aerobic and Anaerobic Respiration

Fermentation: Balances the reduction of NAD+ with a subsequent oxidation of NADH. Restores electrical balance. Net yield: 2 ATP and “waste” products that are derived from the original electron donor.

ATP Synthase Structure & Function F 1 subunits: 3 a, 3 b, 1 g, 1 d, and 1 e. b subunits = site of ATP hydrolysis/synthesis F 0 subunits: proton channel Torque generated rotor stator

F 1 Subunit Topview ATP Synthase acts as a rotary motor turning in 120 degree steps. 3 or 4 protons flowing through turns c proteins enough to change conformation of b protein 1 ATP

Conformation of active sites on b subunits is constantly changing… like watching The Wave at a Mariners game. Loose Open: substrate binding Tight: product release from highaffinity site, actually requires more energy than ATP synthesis

The force that powers ATP Bidirectional: synthase is the proton motive force, or Dp. Major route of H+ into cell, but protons can get pumped out, too. ATP synthase reaction is close to equilibrium; direction depends on Dp One site for ATP hydrolysis and synthesis (Protons can also be pushed back out by reverse e- flow, usually in chemolithotrophs

R- + K+ K+ R - K+ K+ K+ + + - K K+ K+ K+ R K+ K+ + K valinomycin + K+ + K K K+ K+ R R- Uncouplers are ionophores that collapse the Dp. They inhibit ATP synthesis that is coupled to e- transport. Other cations could run ATP synthase too… Judicious choice of ionophores (small lipidsoluble, cation-carrying, ion gradient-thwarting molecules) show that you can run ATPase with a K+ gradient. Valinomycin, added to cells loaded with K+, uncouples Dp by carrying out K+ and creating a temporary diffusion potential.

Net gain …in the rich world of the gut where E. coli lives!

Bacteria e- source Archaea e- sink

A theory on a uniting feature of the Archaea: energetically stressed lifestyle. Archaea are adapted to energy-poor extremes, but Bacteria outcompete them in “easy” environment. Success in Stress: Marine sedimentary record of large radiation of tetraether-based, isoprene membranes in an anoxic period during the mid-Cretacean, perhaps driven by competition for ammonia (e- donor) utilization? Doesn’t prefer life of luxury: Low extant diversity (89 genera vs. 1, 400 bacterial genera) due to inability to radiate in replete environments

Energetic stress: Halophilic lifestyle (must prevent excess Na+ from entering cell) Thermophilic lifestyle (must keep from melting/denaturing) Nitrifiers (low N, low O 2 in open ocean) Anaerobic methanogens (always Archaea) (not a good living: one methane produced can gain as little as one cation (1/3 to ¼ ATP) Aerobic methanotrophs Acidophilic lifestyle Alkaliphilic lifestyle – think what this does to one’s Dp!! -Na+ powered flagellae and ATP synthase -local H+ gradients (proton pump next to ATPase? -S-layers, membrane properties retain H+

Archaea are good at maintaining cells. Bacteria prefer the “growth” lifestyle. Few or no Archaeal pathogens because living in a host is not adapted to a lowenergy ecosystem

Archaea survive heat, low p. H; Bacteria prefer cool and neutral D. L. Valentine, 2007, Nature Reviews Microbiology 5: 316

Membranes: Stable to temperature, p. H Less permeable to ions so less leakage/uncoupling; better maintenance of chemiosmotic potential D. L. Valentine, 2007, Nature Reviews Microbiology 5: 316

Speaking of stress… Fermentation, a very stressful situation O 2 is not highly soluble in water Many environments easily become anoxic Decomposition of organic material must continue anaerobically If alternate e- acceptors (SO 4 -2, NO 3 -, Fe 3+) are not available, then… Fermentation! ~3 ATP per glucose now, not 38 as in aerobic respiration.

Respiration vs. Fermentation ADP + Pi ½ O 2 NADH + H+ B NADH + H+ FADH 2 FAD H 2 O NAD+ ATP NAD+ BH 2 O

Fermentation – Key Features (1) Always anaerobic (if O 2 around, can’t be used) (2) No externally supplied terminal electron acceptor. (3) Substrate-level phosphorylation is the rule*. Many types. . 2 major themes (1) NADH+H+ gets oxidized to NAD+ (2) Electron acceptor is usually Pyruvate or its derivative. *Rules are always meant to be broken!

Pasteur Effect: ~20 X more biomass when aerated

Redox balance

The short list

Fermentation as a diagnostic tool: end products are a metabolic fingerprint Escherichia, Salmonella, Proteus, Aeromonas: Ferment glucose to a number of organic acids. Produce H 2 and CO 2: enzyme called formic hydrogenlyase splits formic acid to make these. Enterobacter makes neutral fermentation products.

Fermentation as a diagnostic tool: end products are a metabolic fingerprint Enteric bacteria: g-proteobacteria, G- rods, nonsporulating, facultative anaerobes, oxidase-negative, fermentative Enterobacter: negative Escherichia, Salmonella, Proteus, Aeromonas: positive Methyl Red Test: Red if p. H < 5. 0

Escherichia, Salmonella, Proteus, Aeromonas: negative Voges-Proskauer Test: pink = butanediol produced Enterobacter: positive

Clostridium spp: endospore-forming, anaerobic rods Ferment amino acids, purines, aromatic compounds/antibiotics Pyruvate

Tricarboxylic acid (TCA) cycle: used in aerobic respiration, and even fermentation! Don’t want NADH or FADH 2 Still need precursors for amino acids etc. Convert from oxidative pathway to reductive pathway (incomplete CAC) Blocked between a-ketoglutarate and succinyl-Co. A, cannot operate in oxidative direction Runs in reverse from OAA to Succinyl Co. A Fumarate dehydrogenase instead of succinate dehydrogenase Reverse arrows for NADH and FADH 2

Propionic Acid Fermentation: incomplete citric acid cycle Pyruvate OAA Lactate Mal Variations in many fermentative microbes Fum Succ Co. A Prop Co. A Methyl. Malonyl Co. A

The unusual fermentation of succinate Sodium ATPase: Uses Na+ gradient …DG 0’ so small (-20. 5 k. J) that cannot even do substrate-level phosphorylation! Coupled to sodium transport; slowly build up “equity” in form of Na+ gradient (Dp)

The unusual fermentation of oxalate H+ ATPase: Uses proton gradient …DG 0’ so small (-26. 7 k. J) that cannot even do substrate-level phosphorylation! Oxalate consumes protons in cytoplasm during conversion to formate. Slowly boosts Dp by making cytoplasm more basic.