It is typically taught that P:O ratios can approach or reach 3.0 for mitochondria, and this is also true for many bacteria. To accomplish such high P:O values, however, requires a modified electron transport chain that pumps more protons per pair of electrons (from NADH to oxygen) than is true for E. coli. This is accomplished by adding a ubiquinol-cytochrome c oxidoreductase (also known as the cytochrome bc complex, since it contains both types of cytochromes) to the electron transport chain. See Figure 12. This complex is the equivalent of mitochondrial "Complex III" and is responsible for oxidation of quinols and reduction of cytochrome c. The H+/e- ratio for this complex is 2. That is, for each ubiquinol oxidized, 4 protons are translocated to the periplasmic space. How is this accomplished?

The cytochrome bc complex has two b hemes, a 2-Fe-2S center (the so-called Rieske Fe-S center, named after Rieske who discovered this center in mitochondria), and a membrane-associated c-cytochrome. The cytochrome bc complex also has two binding sites for quinones: one which binds and oxidizes quinol and which is near the periplasmic surface, and one which binds and reduces quinone near the cytoplasmic surface. The Q cycle operates as follows: quinol binds and one electron is tranferred to cytochrome c, one electron is tranferred to quinone at the quinone reduction site forming a semiquinone anion, and two protons are released to the periplasm. The fully oxidized quinone is released, but the semiquinone at the quinone reduction site is tightly bound. These reactions are repeated: a 2nd cytochrome c is reduced as quinol is oxidized, and the quinone at the reduction site becomes fully reduced to form an anionic quinol. Two protons are picked up from the cytoplasm, forming neutral ubiquinol, which is released. The net reaction is that 2 quinols are oxidized, one quinone is reduced to quinol, and two cytochrome c molecules are reduced. This Q cycle allows 4 H⁺ to be translocated in total for 2 electrons transported to cytochrome c (only one quinol is actually oxidized in the net reaction). Pretty clever amplification, since one would expect only 1H⁺ per electron from quinol oxidation by any simple mechanism.

Bacteria which contain the cytochrome bc complex, in addition to an NADH dehydrogenase and cytochrome oxidase can in principle translocate about 12 protons as NADH is oxidized and oxygen is reduced. In practice, only about 3 ATPs can be synthesized, however, rather than the theoretical 4. A wide variety of bacteria have these more complex electron transport chains, and hence E. coli is somewhat the exception rather than the normal situation in this particular case.

Most are aerobes and oxidize reduced inorganic compounds using O₂ as electron acceptor. There are a few archaea that can oxidized inorganic compounds and use elemental sulfur (S⁰) as an electron acceptor, however.

2H₂ + O₂ ----> 2H₂O

Some Pseudomonas sp., Paracoccus sp., and Alcaligenes sp.

Electrons typically donated to quinone, and NAD(P)H is synthesized by reversing electron transport through the NADH dehydrogenase complex (i.e., protonmotive force is used to reverse the thermo-dynamically unfavorable reduction of NAD⁺ by QH₂.

Rely on HYDROGENASE, a Nickel-containing enzyme to oxidize H₂ gas. Enzyme is O₂-sensitive, and most organisms utilizing O₂ as acceptor prefer microaerophilic conditions.

H₂ oxidation can also occur anaerobically, with SO₄^-2, S⁰, or CO₂ as acceptors. When eubacteria use sulfate (Desulfobacter sp., Desulfovibrio sp.) or CO₂ (Clostridium sp. or Acetobacterium sp.), the products are H₂S or acetic acid; archaea produce H₂S from sulfate, but produce CH₄ (methane) from CO₂. Pyrodictium sp. (extreme thermophilic arachaean) can use S⁰ as acceptor

Eubacteria: Thiobacillus, Thiosphaera, Thiomicrospira, Thermothrix, Beggiatoa

Archaea: Sulfolobus, Acidianus

Some grow at neutral pH, but many can withstand very low pH (they excrete H₂SO₄!).

Fe⁺² also used by those that grow at low pH

Organisms prevalent in PA, since acid-mine drainage contains both reduced S compounds and Fe⁺² iron. Acidification of streams occurs, causing deposition of Fe(OH)₃

Important enzyme is sulfide:quinone oxidoreductase that uses electrons from H₂S to reduce quinones. S⁰ is oxidized to sulfite and finally sulfate.

All are capable of CO₂ reduction (carbon fixation) and are hence "autotrophs." Reduced pyridine nucleotides are formed by "reverse electron transport" using proton gradient to drive reduction of NAD(P)H by QH₂ pool.

Nitrification refers to the oxidative conversion of ammonia to nitrate. Overall process requires two groups of bacteria: the ammonia-oxidizing bacteria = nitrosifyers (e. g., Nitrosomonas, Nitrosococcus) and the nitrite-oxidizing bacteria = nitrifying bacteria (e.g., Nitrobacter, Nitrococcus). Both groups are autotrophs and use CO₂ as carbon source.

NH₃ is first converted to NH₂OH by a monooxygenase, using NADH as electron donor. This step consumes energy. Oxidation of NH₂OH to NO₂^- produces a proton gradient coupled to ATP formation. Most of these organisms can also oxidize CH₄.

Nitrite is oxidized in one step to nitrate, producing sufficient energy to form one ATP. Reduction of pyridine nucleotides occurs by reverse electron transport.

Electron acceptors other than oxygen for electron transport processes--mostly inorganic

Includes: NO₃^- (product NO₂^-)

SO₄^-2 (product H₂S)

Fe⁺³ or Mn⁺⁴ (product Fe⁺² or Mn⁺²)

S⁰ (product H₂S)

CO₂ (products CH₄ and acetate)

fumarate (product succinate)

DMSO, TMAO (products DMS, TMA)

DENITRIFICATION is an anaerobic respiration that uses NO₃^- as electron acceptor when O₂ is absent. Products include NO₂^-, NO, N₂O, and N₂. The latter three are gases that are lost to the atmosphere, hence Nitrogen is lost from soils, etc. when this process occurs.

Common process in a variety of soil bacteria under anaerobic conditions (e.g., Pseudomonas sp.). Energetically less favorable than O₂, but nonetheless rather favorable (see redox potential Table).

SO₄^-2 is less favorable than nitrate, but still often used as an acceptor of electrons from H₂ or organic compound oxidation under anaerobic conditions. Examples: Desulfovibrio, Desulfobacter sp. Sulfate is converted to APS, reduced to SO₃^-2 and then reduced to H₂S and secreted. Protons are translocated as hydrogen is oxidized in the periplasm while SO₄^-2 reduction takes place in the cytoplasm. The H⁺ gradient is used to synthesize ATP.

At low pH (pH 2) Fe⁺³/Fe⁺² has a potential of +770 mV. Reduction of Fe⁺³ to the more soluble Fe⁺² occurs with oxidation of a variety of organic compounds. Solubilization of iron is biologically and geochemically important. Some organisms can also reduce Mn⁴⁺ to Mn²⁺. The ET chains presumably end with ferric iron reductases.

Example: Shewanella putrefaciens

Other metals and metal oxides (e.g., selenate SeO₄^-2 and arsenate AsO₄^-3) can also be reduced by some bacteria.

As noted when considering H₂ as electron source, oxidation of H₂ can be coupled to CO₂ reduction to either acetate or methane.

Acetate can be formed by Clostridium aceticum (G+) or Acetobacterium woodii (G-). Energy yield is about 30% lower than methane production. ATP is formed from substrate-level phosphorylation (Acetyl-P donates ~P to ADP at last step of path) and H⁺/Na⁺ gradient.

Interesting point: C. aceticum produces H⁺ gradient during acetogenesis. However, A. woodii produces a Na⁺ gradient, and then couples the Na⁺ gradient to H⁺ translocation for ATP synthesis.

1-C unit is reduced to -CH₃ after attachment to tetrahydrofolate, then transferred to Vit. B₁₂

2nd CO₂ is reduced to CO on the Ni-Fe enz. carbon monoxide (CO) dehydrogenase. This enzyme also combines the -CH₃ group from methyl-Vit. B₁₂ with carbonyl group and CoA to form Acetyl-CoA, which ultimately can form Acetyl-P for ATP formation.

Methanogens are members of the Archaea, and only Archaea exhibit the ability to form CH₄

Methanogens also use the Acetyl-CoA pathway for biomass production, but -CH₃ groups come from the CH₄ production pathway (i.e., they use CO dehydrogenase ). Process is similar in that CO₂ is first reduced to Formyl on a special coenzyme called methanofuran, then transferred to tetrahydromethanopterin, a 2nd novel coenzyme where the formyl group is reduced to the -CH₂^- level, then the -CH₃ level. The -CH₃ group is transferred to a 3rd coenzyme, CoM-SH, forming
Co-M-CH₃. Finally, methyl reductase reduces the methyl group, releasing CH₄

The last step produces a proton gradient; cleavage of methyl bond is achieved with production of Co-M-S-S-HTP (HTP-SH is another unique coenzyme: 7-mercapto- heptanoylthreonine) disulfide bond. A heterodisulfide reductase reduces the mixed disulfide and forms a proton gradient. Hydrogenase releases H⁺ outside the cell as well, but H₂ concentrations very low, and energy yield is approximately 1 ATP per mole of CH₄ formed! This is a lousy way to make a living, so to speak.

E. coli and other bacteria can use some organic compounds as electron acceptors under anaerobic conditions. Fumarate reductase transfers electrons from menaquinol (MQH₂)to fumarate, producing succinate which is excreted. Fumarate is less favorable than nitrate as an acceptor, and NO₃^- suppresses the expression of the genes for fumarate reductase. NADH coupled to fumarate reductase produces ~1 ATP per NADH consumed. DMSO and TMAO are energetically more favorable, and their oxidoreductases do translocate H⁺.

Critical features are that organic compounds serve as sinks for NAD(P)H electrons and redox balance must be maintained. ATP synthesis by substrate-level phosphorylation only.

Fermenations are "rearrangement" reactions. Substrate(s) of intermediate oxidation status are rearranged to more stable products; part of substrate(s) is more oxidized, part more reduced. Energy yields are low: 1-3 ATP per mole substrate maximum!

Molecules that can be fermented: sugars, most amino acids (not by all species, however), and organic acids.

Little carbon can be siphoned off for biosynthesis, so there is large substrate consumption/conversion with little biomass formation. In E. coli, only ~10% of substrate converted to biomass.

Why the diversity of products? Allows some flexibility with respect to substrates--by adjusting the amount of products, compounds more or less oxidized than glucose can be accomodated (i.e., the ratio of products for glucose would be different for growth on sorbitol (more reduced than glucose) and glucuronic acid (more oxidized than glucose).

Fermentations are industrially important (see handout) due to production of commercially important compounds (acetone, butanol, isopropanol, etc.) and for cheese production, alcohol production etc. See handout for various products made during fermentations.

PHOTOTROPHY means light is the energy source.

Light can provide energy by two fundamentally different mechanisms.

1. Light-activated proton extrusion.

2. Light-activated electron transport with secondary proton extrusion.

The first mechanism is found in some members of the Archaea, namely the Halobacterium sp. Halobacterium and Natronococcus sp., and relatives (about 8 genera) are mostly obligately aerobic chemoorganotrophs. Prefer organic acids and amino acids over sugars in general as carbon/electron/energy sources. These bacteria are extreme halophiles (1.5-5.5 M NaCl; optimal growth at 2-4 M NaCl). Some species also extreme alkaliphiles (pH optimum about 9.5).

Halobacterium sp. synthesize a membrane protein, BACTERIORHODOPSIN, under anaerobic conditions. This protein carries retinal, the same RETINAL (derived from vitamin A) as found in the vertebrate eye. Protein is also somewhat similar to rhodopsin of the eye.

So-called "PURPLE MEMBRANE" is formed: about 25% lipid and 75% bacteriorhodopsin.

Retinal is covalently bound to the -NH₂ group of a Lysine residue; light-absorption cause isomerization of the retinal with resulting release of a proton to the periplasm. In the dark, the retinal re-isomerizes back to original configuration and bind a proton from the cytoplasmic side of the membrane. In this cycle, light-drive proton extrusion from cytoplasm to periplasm results. A related protein, HALORHODOPSIN, can transport chloride (Cl^-) into the cell. In these proteins, retinal acts essentially as a light-activated gate that allows the specific passage of a single ion (H⁺ or Cl^-) per cycle.

Bacteriorhodopsin and ATP synthase can be artificially inserted into phospholipid vesicles. Such vesicles were used by Racker and Stoekenius to provide strong support for the chemiosmotic coupling hypothesis.