Archebacteria and Energy Generation
The archebacteria are related only distantly to the other bacteria. Comparison of 16S ribosomal RNA sequences shows that the archebacteria are related to each other but not to eubacteria or eukaryotic cytoplasm. In fact, there is as much genetic distance between the archebacteria and the eubacteria ("true bacteria") as between the eubacteria and the cytoplasmic component of eukaryotic cells.
The archebacteria have certain biochemical features in common. In particular, their lipids do not have ester-linked fatty acids. The membrane consists of a bilayer of long chain isoprenoid hydrocarbons joined at the ends by ether linkages to glycerol. The head group may be phosphate or contain sugars. Some double-length isoprenoid hydrocarbon chains stretch across the whole membrane. In addition the cell wall contains no peptidoglycan.
Today the archebacteria are found mostly in extreme environments. We will consider the halobacteria and the methanogens, both of which have unique ways of generating energy.
Some Examples of Archebacteria:
Methane Producers. Obligate anaerobes which are very sensitive to oxygen. Convert H2 + CO2 ===> H2O + CH4. Metabolism is unique - they contain coenzymes found in no other living organisms. They have no cytochromes, flavins or quinones.
Sulfolobus. Optimum pH 2-3, temperature optimum 70-80°C. Lives in geothermal springs, etc. and oxidizes sulfur to sulfuric acid. Various other sulfur metabolizing Archebacteria also exist.
Thermoplasma. No cell wall. Optimum growth temperature 55°C, optimum pH about 2.
Methane Production
This is a unique property of the methane bacteria - one class of Archebacteria. Habitat must be anaerobic with redox potential more negative than -200mV. Not only must oxygen be absent but so must other electron acceptors such as nitrate. Furthermore, if sulfate and sulfate reducing bacteria are present these will consume any H2 which is available in preference to the methane producers. Many methane bacteria are found in the rumen of cows where they produce methane from the H2 and CO2 released by other anaerobic gut bacteria.
Methane production is NOT a fermentation. There is no substrate level phosphorylation and ATP is generated via the PMF. However, the mechanism is still uncertain. Furthermore, since there are no cytochromes, flavins or quinones the electron transport chain must be quite different. About 90-95% of the CO2 is converted to methane and the energy derived is used to fix the remaining CO2 into cell material.
4H2 + CO2 = CH4 + 2H2O Delta G° = -31 kcal/mole
In natural habitats the concentration of H2 is very low, at most one micromolar and the actual Delta G is approximately -15kcal/mole. Growth yields indicate one ATP per CH4 as would be expected for a cell which is around 50% efficient (as most are).
Methanogenesis and its Cofactors
Metabolism involves a hydrogenase which uses H2 gas (E' = -420mV) to reduce a unique coenzyme, F420, which has an E' = -380mV. F420 fluoresces blue at 420nm and allows methanogens to be identified in the fluorescence microscope. Other organisms do possess F420 derivatives used in DNA repair. However only methanogens contain enough F420 to give the cells a blue flourescence.
Coenzyme F420 structure:
Reduced F420 plays two roles:
b) Transfer of 2[H] to the methane system. The 2[H] cannot go via NAD or NADP as their E' is only -320mV, not negative enough for making methane. Four lots of 2[H] are required for each CH4 produced.
F420 differs from FAD in three respects (see diagram):
b) N at bottom of centre ring in FAD is replaced by CH in F420
c) R-group is ribitol-phosphate-phosphate-ribose-adenine in FAD but ribitol-phosphate-lactyl-glutamate-glutamate in F430:
The methane pathway involves several unique coenzymes, some of which carry the one carbon unit during its 4-step reduction to methane:
a) Methanofuran (MFR) picks up the CO2 and carries it for the first reduction step which gives formyl-MFR.
The R-group has the structure:
b) Tetrahydromethanopterin (THMP) carries the one carbon unit during the next two reductions. THMP is a pterin derivative related to folic acid which carries one carbon fragments during biosynthesis. Do not pronounce the p.
c) The one carbon unit is held by coenzyme M during its reduction to methane in the final step.
d) Factor B = HTP-SH (7-mercapto-heptanoyl-threonine phosphate) reacts with methyl CoM and the disulfide CoM-S-S-HTP is formed.
e) The methyl reductase step requires the cofactor F430 which is a Nickel containing tetrapyrrole needed for electron transfer.
Energy production occurs in two places. Firstly, the reduction of the disulfide CoM-S-S-HTP back to the separate -SH compounds, by an FAD-containing heterodisulfide reductase, generates energy as proton motive force. Secondly, the transfer of the CH3 group from CH3-THMP to CoM results in energy release but generates a sodium gradient, not a proton gradient. Apparently there are two ATP synthases in methanogens, one coupled to the proton gradient the other using the sodium gradient.
Other one carbon compounds (e.g., HCOOH, CH3OH) may be converted to methane by certain methane bacteria. In addition Methanosarcina and some other methanogens can convert CH3COOH Æ CH4 + CO2 by reversing some of the reactions of the biosynthetic the "carbon monoxide reductase" pathway. In nature, up to 70% of the methane produced may come from acetate (rather than free CO2).
It was originally wrongly thought that Coenzyme B12 was involved in methane production because if Coenzyme B12-CH3 is added to cell extracts it will donate the -CH3 to CoM. However, it will also donate its -CH3 group to almost anything else in vitro.
Although CoB12 is not actually involved in methanogenesis it is involved in the synthesis of cell material by methanogens. Methanogens do not possess the Calvin cycle. The major route for incorporation of CO2 into cell material is via the "carbon monoxide reductase" pathway (see diagram). One carbon dioxide is reduced to a methyl group by the methane pathway and transferred to Coenzyme B12. Another carbon dioxide or carbon monoxide is coupled with the CH3 group to give an acetyl group. CODH is carbon monoxide dehydrogenase.
Acetyl-CoA is converted to other metabolic intermediates. The critical steps are the use of reduced ferredoxin (FdH2) to drive the decarboxylation of pyruvate and of alpha-ketoglutarate backwards:
(i) FdH2 + CH3CO-SCoA = CH3COCOOH + CoASH + Fd
(ii) FdH2 + Succinyl-CoA = alpha-ketoglutarate + CoASH + Fd
Interspecies Hydrogen Transfer
A fermenting organism must reoxidize its H-carriers (usually NADH), hence producing fermentation products. An alternative exists - this is the production of molecular H2. In practice this can only occur if either the redox potential of the reaction is nearly as negative as H2 (e.g. CO2/HCOOH and 2H+/H2 both have E' = -420mV so that formate dehydrogenase can release H2 and CO2), or if the H2 is removed so pulling the reaction over. This may be done artificially by flushing a culture with an inert gas or by adsorbing the H2 onto palladium. Biologically it can be done by adding methane producing bacteria.
In nature, fermenting bacteria make substantial gains in energy efficiency and carbon usage by dumping their H2 onto methane bacteria. Below a partial pressure of H2 of 1/1000 atm it becomes energetically favorable to convert NADH to NAD plus H2. In practice only those organisms which can couple NADH to a carrier called ferredoxin are able to take advantage of low H2 pressure. Ferredoxins are non-heme iron proteins of low molecular weight (54 amino acids in Clostridium). Those involved in fermentation usually have Fe4S4 centers.
In natural habitats we have four main stages in fermentation:
b) Clostridium, Selenomonas, Ruminococcus etc. (i.e. obligate anaerobes) when grown alone produce ethanol, lactate, etc. depending on the species. If methanogens are present they can send 2[H] from NADH to ferredoxin and then to H2 gas and are no longer forced to produce ethanol and lactate but can excrete the more highly oxidized acetate so gaining extra energy. In some of these organisms the oxidation of pyruvate to acetyl-CoA is directly coupled to ferredoxin without the intervention of NAD. However, the 2[H] from glycolysis is carried by NAD at first, and then transfered to ferredoxin.
c) Certain bacteria are capable of taking up and using ethanol (or lactate) under anaerobic conditions. This contrasts with group (b) who can re-route their metabolism to avoid ethanol production -- quite a different matter. Fermentation of ethanol to acetate plus 2H2 can only occur when methanogens are present to remove the H2. An example is the S-organism. This can grow in two ways:
2) Ethanol is fermented to acetate plus 2H2 only if a methanogen is present to consume the H2.
d) Methanogens consume the H2 and the CO2. Although they provide the final step in the fermentation of other organisms, methanogens don't themselves ferment. Methanogenesis is a strange form of anaerobic respiration.
Breakdown of acetate anaerobically occurs by three known routes:
2) Acetate ==> 2CO2 by Desulfuromonas using sulfur as electron acceptor.
3) Acetate ==> CH4 + CO2 by Methanosarcina and some other methanogens.
Note that in the absence of sulfate and in the presence of a methane producer Desulfovibrio can ferment ethanol to acetate (or lactate to pyruvate) plus H2 and thus can belong to group (c).