Most hydrogenosomes are spherical or slightly elongated granules, with a homogeneous particle matrix inside. However, in some cells, the hydrogenosomes are not spherical but are very elongated, such as those found in Monocercomonas sp. ^[8]. The hydrogenosomes found so far are encased by two adjacent membranes, which are very thin, presenting a thickness of only a few nanometers ^[9]. Although researchers have reported that the hydrogenosome of Neocallimastix sp. L2 is monomembraned ^[10], Benchimol et al. modified the traditional fixation procedure with CaCl₂ and, through transmission electron microscopy and frozen sections, found that the inner and outer membranes are very close to each other ^[9]. This indicated that an appropriate fixation method is necessary for good visualization of the two membranes. In addition, researchers have found that in some regions of the hydrogenosome of Trichomonas foetus (T. foetus), the separation between the two membranes forms a vesicle structure. Under transmission electron microscopy, the electron density of the vesicles was slightly higher than that of the matrix ^[11]. The number, size, and electron density of the vesicles varied in different microorganisms ^{[12][11][12][13][14]}. These vesicles may be related to the storage of calcium and other cations and change at different metabolic states.

In 1973, Lindmark and Muller discovered that the organelles in T. foetus did not possess the typical mitochondrial characteristics of the tricarboxylic acid cycle, electron transport chain, F₀–F₁ ATPase, and oxidative phosphorylation, but the enzyme system in the particle matrix was capable of completing the anaerobic metabolism of pyruvate. In contrast to mitochondria and peroxisomes, these organelles directly use protons as terminal electron receptors to produce H₂; therefore, they proposed a name for this subcellular particle called hydrogenosome in conjunction with previous studies ^[13]. The hydrogenosome converts pyruvate into various metabolites, such as formate, acetate, H₂, and CO₂, and also produce ATP through substrate-level phosphorylation for cell growth, acting as an oxygen-independent mitochondrion.

In addition, the hydrogenosome possesses another important function for its survival known as antioxidative stress response. The enzymes in the hydrogenosome, especially pyruvate: ferredoxin oxidoreductase (PFO) and hydrogenase, are highly sensitive to O₂ and can be inactivated rapidly under the condition of O₂, blocking the substance metabolism in the hydrogenosome. The inhibition of the enzyme by O₂ may be due to the formation of reactive oxygen species (ROS). For example, O₂ is thought to bind to [Fe] at the far end of the [2Fe]H subcluster, then form an ROS and destroy the [4Fe4S]H subcluster to inactivate the [FeFe] hydrogenase ^[15]. In most living organisms, iron-dependent superoxide dismutase (SOD) converts ROS to hydrogen peroxide (H₂O₂), which is subsequently removed by mitochondria usually using the glutathione system and catalase. The mechanism by which the hydrogenosome defends against the damage from ROS is not entirely the same. Although Lindmark et al. reported SOD activity in T. foetus hydrogenosomes ^[16], there have been no reports of peroxide reductase in the organelles that break down H₂O₂. Unlike mitochondria, thioredoxin-linked peroxiredoxin antioxidant system is one of the major antioxidant defense mechanisms in trichomonas hydrogenosomes. Three relevant peroxidases in the hydrogenosome of Trichomonas vaginalis (T. vaginalis), thioredoxin reductase (TrxR), thioredoxin (Trx), and Trx-dependent peroxidases (TrxP) jointly play a role in the hydrogenosome to reduce oxidative stress caused by H₂O₂. Moreover, when T. vaginalis is subjected to oxidative stress, the expression of Trx and TrxP is increased at both transcriptional and translational levels, demonstrating that the parasite is capable of responding to exogenous oxygen stress through changes in both levels ^[17].

Due to the difference in structure and enzyme, the hydrogenosomal metabolism in different microorganisms is not exactly the same. In general, there are many enzymes related to carbohydrate metabolism in the hydrogenosome, including malic enzyme (ME), PFO, [2Fe-2S] ferredoxin protein (Fdx), succinate thiokinase (SCS), adenosine kinase (AK), [Fe] hydrogenase (Hyd), acetate: succinyl CoA-transferase (ASCT), among others. Glucose enters the glycolysis process when it is transferred into the cytoplasm by glucose transporters. Then the formed malate or pyruvate goes into the hydrogenosome; it is metabolized as CO₂, formate, and acetate and finally releases energy in the form of ATP. PFO and hydrogenase are two hydrogenosomal enzymes that do not exist in mitochondria. Instead of the pyruvate dehydrogenase enzyme complex, PFO converts the pyruvate produced by glycolysis to acetyl CoA and CO₂ (by taking off the hydroxyl group). Then the consequent electron is passed to the middle electronic carrier Fdx, and the reduced Fdx is oxidized by ferredoxin hydrogenase, eventually passing electrons to protons (H⁺) to form H₂. H₂ in the hydrogenosome can also be produced in a determined way by hydrogen dehydrogenase; however, the enzyme mainly operates the reverse reaction to converse NAD(P)⁺ and H₂ to H⁺ and NAD(P)H, which is in an energetically favorable direction. In addition, a speculative way of bifurcating hydrogenase to produce H₂ has been put forward and seems important in the coupling of the reduction of H⁺ to the oxidation of NAD(P)H through the ferredoxin to generate H₂ ^[18]. ATP is formed by substrate-level phosphorylation ^[19][20] (Figure 3).

Carbohydrates are the first source of energy for a living organism. Living organisms will absorb other nutrients, such as amino acids, to sustain its growth when carbohydrates are insufficient. Previous studies have shown that under normal culture conditions, T. vaginalis consumes large amounts of arginine and small amounts of methionine for energy production; and when maltose is deficient, it consumes higher amounts of amino acids, especially arginine, threonine, and leucine ^[21].

Mukherjee et al. reported the identification and analyses of two glycine cleavage H proteins and a dihydrolipoamide dehydrogenase (L protein) from T. vaginalis, which are pivotal members of the glycine decarboxylase complex, which catalyzes the oxidative decarboxylation and deamination of glycine. They determined their location in the hydrogenosome by immunofluorescence analysis, indicating the important role of the hydrogenosome in amino acid metabolism ^[22]. The amino acid synthesis and decomposition are associated with the Krebs cycle, glycolytic pathway, and pentose phosphate pathway through conversion to intermediate products (such as pyruvate, acetyl CoA, oxaloacetic acid, acetoacetic acid) in the glucose metabolism in the mitochondria. Similarly, metabolism of amino acids is linked with glycolysis for ATP production in the hydrogenosome. Many enzymes associated with amino acid metabolism and other related metabolic pathways in the protein metabolism at the gene level have been identified ^[23]. In addition to using immunology at the protein level, or RNA sequencing at the transcriptional level to analyze the differential expression of genes under changing environmental conditions, Huang et al. used LC–FTMS for the first time to analyze the regulation of amino acid metabolism in the hydrogenosome of T. vaginalis and its adaptive response in a glucose restriction environment at the metabolome level. The results showed that amino acid metabolism in the hydrogenosome of T. vaginalis was significantly altered in the case of carbohydrate deficiency. The endergonic metabolism of methionine was less stimulated to reduce ATP consumption, while the branched chain amino acid synthesis related to the glutamate metabolic pathway was markedly enhanced for energy generation ^[24].

The findings on the metabolism of the hydrogenosome are mainly based on the results of experiments conducted in the past several decades using trichomonas as a model. Therefore, the conclusions cannot prove that all hydrogenosomes have the same enzymes and metabolic activities. In fact, the enzyme composition and material metabolism in the hydrogenosome of anaerobic fungi are different from that of other microorganisms (Figure 35). Unlike other anaerobic microorganisms, anaerobic fungi depend on pyruvate: formate lyase (PFL), which exists in both the cytoplasm and the hydrogenosome to hydrolyze pyruvate into isomole formate and acetyl CoA, rather than PFO. Though many studies have suggested that PFO is either absent or of only marginal importance in the anaerobic fungal hydrogenosomal metabolism, both enzymes were identified in all published gut fungal genomes, and a metabolic model proposed and analyzed by Wilken et al. suggested that PFL carries more metabolism flux than PFO in the hydrogenosome, but an energetically favorable route to H₂ production still requires the action of PFO ^[18]. The pyruvate in the cytoplasm is eventually metabolized to ethanol, and the pyruvate that enters in the hydrogenosome is eventually metabolized to acetate. Pyruvate can be directly transported into the hydrogenosome, and also may be formed from the dehydrogenation of malate entered into the hydrogenosome. CO₂ and NAD(P)H are also produced during the dehydrogenation of malate, and then hydrogenase reduces 2H⁺ to H₂ with or without the reducing equivalent of NAD(P)H. Acetyl-CoA formed in the hydrolysis of pyruvate first generates acetate and succinyl-CoA under the action of succinyl-CoA transferase. Then succinyl-CoA is converted to succinate by succinyl-CoA synthase, along with the release of CoA and conversion of ADP to ATP, which supplies energy for anaerobic fungi. Therefore, PFL is one of the most important enzymes in the sugar metabolism of anaerobic fungi. Thus, in addition to H₂, CO₂, acetate, lactate, ethanol, and succinate, the fermentation products of carbohydrates in anaerobic fungi contain formate in equal molar quantities with acetate plus ethanol ^[25].

So far, there have been no reports on the metabolism of amino acids in the hydrogenosomes of anaerobic fungi.