Stealth reactions driving carbon fixation

Organisms live in an interconnected dynamic web in which they make, degrade, and interconvert compounds containing carbon, hydrogen, nitrogen, oxygen, phosphorus, and sulfur. The carbon cycle involves the oxidation of organic compounds to produce CO₂ by heterotrophic organisms and the incorporation (“fixation”) of CO₂ from the environment into living tissue by autotrophic organisms. Heterotrophic organisms (most animals) obtain the energy for life by conserving the energy obtained by oxidizing organic molecules to CO₂ in the form of reducing equivalents (electrons) and adenosine triphosphate (ATP). Autotrophic plants, bacteria, and archaea fix CO₂ by a process in which the energy of electrons and ATP is used to produce biomolecules, such as sugars, amino acids, and lipids, thereby replenishing these essential organic molecules in the ecosystem. Cumulatively, autotrophy occurs on the huge scale of 7 × 10¹⁶ g of carbon fixed annually (1). Six CO₂ fixation pathways differing in their ATP requirements are known to exist. Fixing CO₂ using the least ATP possible is key for anaerobes because their metabolism generates much less ATP than does growth on oxygen. The reductive tricarboxylic acid (rTCA) cycle is one of the most evolutionarily ancient and least ATP-demanding autotrophic pathways. On pages 563 and 559 of this issue, Mall et al. (2) and Nunoura et al. (3), respectively, uncover an unexpected ATP-conserving mechanism, and Pachiadaki et al. (4) report a surprising source of reducing equivalents in the rTCA cycle.

According to our understanding of the rTCA cycle, three enzymes that catalyze reactions in the oxidative TCA (oTCA) cycle that would be highly thermodynamically unfavorable if performed in the reverse direction are substituted by parallel energetically favorable reactions (5) (see the figure). In the rTCA cycle, citrate synthase (CS), which catalyzes conversion of oxaloacetate and acetyl-CoA (coenzyme A) to citrate and CoA, the most irreversible reaction in the oTCA cycle, is replaced by ATP-dependent citrate lyase (ACL) or homologous enzymes that catalyze this reaction in two steps (5, 6). The existence of ACL is the typical indicator for an operational rTCA cycle in an organism (2). The use of ATP in the ACL reaction converts a mountainously thermodynamically unfavorable reaction into a slightly uphill one. Thus, the direct physiological reversal of CS has been thought to be impossible.

The autotrophic roTCA cycle.

In the rTCA cycle, two CO₂ molecules are fixed to generate acetyl-CoA, which is used for biosynthesis. Because three reactions in the oTCA cycle are irreversible, they are substituted by ATP-dependent enzymes, such as ACL, in the rTCA cycle. Surprisingly, in the roTCA cycle, citrate synthase (CS) is retained. The discovery of nitrite oxidation as a source of electrons in the rTCA cycle is another unexpected turn of events in carbon fixation.

It is time to change these views of CS and of the rTCA cycle. Mall et al. and Nunoura et al. showed that two thermophilic sulfur-reducing anaerobic bacteria, Desulfurella acetivorans and Thermosulfidibacter takaii, lack the gene encoding ACL, but retain all other rTCA cycle enzymes. Both organisms also lack the genes required for other known CO₂ fixation pathways. Surprisingly, despite the high thermodynamic barrier, they found that CS functioned in reverse. To distinguish this pathway from the typical rTCA cycle, this was termed the reversed oxidative TCA (roTCA) cycle.

How do these organisms overcome the huge thermodynamic barrier for reversing CS? Apparently, they rapidly and efficiently couple roCS with the succeeding reaction(s) to keep the cellular roCS substrate (citrate and CoA)/product (oxaloacetate and acetyl-CoA) ratio extremely high. Mall et al. detected very high activities of both roCS and reverse oxidative malate dehydrogenase (roMDH), which would promote rapid conversion of oxaloacetate (from roCS activity) to malate. However, enhancing the activity of these enzymes will increase the rates of both oxidative and reverse reactions. The key is flux. These organisms must efficiently convert the roCS and roMDH products malate and acetyl-CoA into cellular biomolecules, thus maintaining very low concentrations of oxaloacetate, effectively pulling flux through the roTCA cycle. Even by bypassing the ATP provided by the ACL-catalyzed reaction in the rTCA cycle, the overall roTCA cycle is thermodynamically favorable; thus, another trick may involve bifurcation, driving the unfavorable half of the cycle by coupling it to the favorable half, yielding a net spontaneous process. Nunoura et al. suggest that abundant input of redox equivalents via electron bifurcation may provide the needed thermodynamic push. However, roCS is not an electron transfer reaction, so it would require an unknown mechanism to make such a bifurcation work. To add to the mystery, the CSs of both organisms have rather typical kinetic parameters and are not specifically adapted to run in reverse.

The essential point is that reversing CS spares one ATP for the organism during autotrophic growth, which is potentially important as ATP is of premium value for anaerobes. Thus, these fastidious microbes require a single ATP to fix two molecules of CO₂, matching the stoichiometry, efficiency, and free energy (a measure of thermodynamics) of the Wood-Ljungdahl pathway (6). By contrast, the prevalent CO₂-fixing pathway, the Calvin cycle, uses seven ATPs. Not surprisingly, the Calvin cycle has not been found to occur in anaerobes. In aerobes, O₂ is used to generate an ample supply of ATP. Furthermore, because ACL is the signature enzyme for detecting the rTCA cycle, the roTCA cycle could be a widespread mode of CO₂ fixation, previously hidden below our genomic radar.

Mall et al. asked if there are other examples of unexpected reactions running in reverse. Anaerobic methane oxidation is one example. This is an unusual pathway that is important to the carbon cycle in which the entire reaction sequence of CO₂ reduction to methane runs in reverse (7). This appears to occur in a microbial community by coupling the reducing equivalents generated by the methane oxidizer to the highly favorable reduction of electron acceptors—for example, sulfate—by a sulfate-reducing organism (8).

A mode of CO₂ fixation by rTCA based on nitrite oxidation has also been lurking unnoticed in the dark ocean where light is too dim to produce ATP using photosynthesis and fix CO₂ by the Calvin cycle. Using metagenomic analyses of nitrite-oxidizing bacteria, Pachiadaki et al. identified a highly represented phylum, Nitrospinae, which contains genes encoding nitrite oxidoreductase and rTCA cycle enzymes, yet lacks the genes for other energy-generating pathways such as ammonium or sulfur oxidation. Thus, CO₂ fixation in the rTCA cycle by these bacteria appears to rely on nitrite oxidation as a source of electrons. The authors found that nitrite oxidation appears to be responsible for up to 43% of the total carbon fixed at some ocean depths. This process has thus far been underappreciated because nitrite is found at very low concentrations in the ocean. Correspondingly, the nitrite oxidoreductases in these bacteria have very high affinity for their substrates.

Coming back full circle to the roTCA cycle, the genome of the most frequently encountered marine nitrite oxidizer contains coding sequences with similarity to those of known ACLs (9), making it likely that Nitrospinae use the rTCA cycle to fix carbon, using nitrite as an electron source. However, given the precedent described by Mall et al. and Nunoura et al., the use of roCS in this phylum of bacteria cannot be excluded.

It is exciting to alter our conceptions about the rTCA cycle, a key pathway in the global carbon cycle. rTCA has been studied for 50 years. This pathway and the Wood-Ljungdahl pathway (6) have the lowest energetic costs and are considered to be the most evolutionarily ancient modes of CO₂ fixation. The new findings highlighted here reveal an unusual mechanism (roCS) that microbes use to lower the ATP requirement for the roTCA cycle and the use of a surprising source of electrons (nitrite) to drive the rTCA cycle. Future studies could reveal the details of how such efficient flux through the roCS reaction is accomplished. In addition, we can look forward to results that clearly quantify the relative global contributions of the various pathways and electron sources that drive CO₂ fixation and to the discovery of other autotrophic metabolic systems.