Proton-Pumping Type I NADH Dehydrogenase and Non-Proton-Pumping Type II NADH Dehydrogenase
The major entry point is the transfer of electrons from NADH oxidation to quinone reduction (e.g., ubiquinone or menaquinone). In bacteria, three different types of respiratory NADH dehydrogenases have been identified and characterized on the basis of reaction mechanism, subunit composition, and protein architecture (
11). These include the proton-pumping type I NADH dehydrogenase (NDH-1, complex I), non-proton-pumping type II NADH dehydrogenase (NDH-2), and sodium-pumping NADH dehydrogenase (NQR). Weinstein et al. identified genes for two classes of NADH:menaquinone oxidoreductases in the genome of
M. tuberculosis (
12) (
Table 1). NDH-1 is encoded by the
nuoABCDEFGHIJKLMN operon and transfers electrons to menaquinone, conserving energy by translocating protons across the membrane to generate a PMF (
Fig. 2). The second class is NDH-2, a non-proton-translocating type II NADH dehydrogenase that does not conserve energy and is present in two copies (Ndh and NdhA) in
M. tuberculosis (
12) (
Table 1). Sodium-pumping NADH dehydrogenase has not been reported in mycobacterial genomes.
NDH-1 is composed of 14 subunits (
nuoA-N), which represent a 15,704-bp operon in
M. tuberculosis (Rv3145 to Rv3158) (
Table 1). NuoB, C, D, E, F, and G are peripheral membrane proteins located in the cytoplasmic side, while NuoA, H, J, K, L, M, and N are in the membrane section of the complex with multiple predicted transmembrane domains (from 3 to 16). In contrast, the
nuo operon has been lost from the genome of the intracellular parasite
Mycobacterium leprae except for a single remaining
nuoN pseudogene (
13). NDH-1 uses flavin mononucleotide (FMN) and iron-sulfur clusters to transport electrons from NADH to the quinone pool (menaquinone). The release of the two electrons during the NADH oxidation produces enough energy to pump four protons across the membrane to generate a PMF (
Fig. 2). In
M. tuberculosis, the
nuo operon is essential for neither growth nor persistence in an
in vitro Wayne model (
6).
Mycobacterium smegmatis also contains genes for a type I NADH:menaquinone oxidoreductase (
nuoA-N). However,
M. smegmatis NDH-I activity is very low, representing about 5% of the NDH-2 activity (
14,
15). Rather, increased expression (15-fold) of the
nuo operon in
M. smegmatis was observed in a carbon-limited chemostat in response to a slowdown in growth rate (
16). In the slow-growing
M. tuberculosis and
Mycobacterium bovis strains
, NDH-1 activity is 28 to 50% lower than the NDH-2 activity when measured in membrane fractions of mycobacteria (C. Vilchèze and W. R. Jacobs, Jr., unpublished data), suggesting that the major NADH oxidizing activity is mediated by NDH-2. This is supported by the observation that NADH oxidation by mycobacterial membranes is relatively insensitive to complex I inhibitors (
12). Notwithstanding this,
M. tuberculosis mutants lacking only one of the subunits of NDH-1,
nuoG, have an
in vivo phenotype, where this gene was critical for host macrophage apoptosis inhibition and mouse virulence (
17). This implies that
nuoG and potentially other subunits of NDH-1 are anti-apoptosis factors and are attractive candidates for vaccine development.
Several studies have reported that
nuo is downregulated in
M. tuberculosis during mouse lung infection (
18), during survival in macrophages (
19), in both nonreplicating persistence (NRP)-1 (1% oxygen saturation) and NRP-2 (0.06% oxygen saturation) relative to aerated mid-log growth (
18), and upon starvation
in vitro (
20). The transcription of NDH-2
(
ndh) is also downregulated in
M. tuberculosis during mouse lung infection, but transcript levels for
ndh peak during NRP-2
in vitro, demonstrating that the pattern of
ndh regulation is different between
in vivo and
in vitro conditions (
19). These data are in contrast to
Escherichia coli, in which NDH-1 is usually associated with anaerobic respiratory pathways (e.g., fumarate) and noncoupling dehydrogenases such as NDH-2 are synthesized aerobically (
21).
The non-proton-translocating NDH-2 is a small monotopic membrane protein (50 to 60 kDa) that catalyzes electron transfer from NADH via FAD (noncovalently bound redox prosthetic group) to quinone. No tertiary structural information exists for either the bacterial, plant, or protist NDH-2 enzymes, but the yeast NDH-2 structure was recently solved by two laboratories (
22,
23). NDH-2 is widespread in bacteria and the mitochondria of fungi, plants, and some protists. In some cases more than one copy is present (
24). The role(s) of multiple type II NADH dehydrogenases in prokaryotes, plants, and parasites is unclear. In some organisms with multiple type II NADH dehydrogenases, one copy appears more essential than the other, pointing to nonredundant functional differences (
25,
26).
M. tuberculosis harbors two copies of NDH-2 (
ndhRv1854c and
ndhA Rv0392c) (
12) (
Table 1), which are well conserved among slow-growing mycobacterial species. In
M. tuberculosis, Ndh (1,392 bp) and NdhA (1,413 bp) share 65% identity; however, the FAD and NADH binding motifs,
G21S
GFG
G26 and
G177A
GPT
G182, are highly conserved. Both proteins contain one transmembrane domain located at the amino acids 385 to 407 and 387 to 409, respectively. The Ndh and NdhA proteins of
M. tuberculosis have been shown to be functional NADH dehydrogenases that transfer electrons to the quinone pool via a ping-pong reaction mechanism (
27). NdhA is not present in
M. smegmatis, yet the level of NADH oxidation by
M. smegmatis NDH-2 is several-fold higher than in
M. tuberculosis or
M. bovis and represents 95% of the total NADH oxidation measured (
15). This might correlate with higher NAD
+ concentrations in
M. smegmatis compared to
M. bovis (three times higher, as shown in reference
15). In addition, when
M. smegmatis and
M. bovis were transformed with a replicative plasmid containing
ndh, the ratio NAD
+/NADH doubled (
15), further confirming the involvement of
ndh in the oxidation of NADH into NAD
+.
Several studies have suggested that
ndh is essential for growth of
M. tuberculosis (
12,
26,
28,
29) (
Table 1). Mutations in the
ndh gene of
M. smegmatis result in a pleiotropic effect: temperature sensitivity, amino acid auxotrophy, and resistance to the first-line anti-TB drug isoniazid (INH) and its analog and second-line anti-TB drug ethionamide (ETH) (
14,
15). The
ndh mutants had decreased NADH oxidase activity and increased NADH concentration (
14,
15). Selection for
ndh mutants grown on rich media (Mueller Hinton) led to the isolation of
ndh mutants that were auxotrophic for serine and glycine; this auxotrophy was resolved with complementation by a wild-type copy of
ndh. The correlation between
ndh mutations and serine/glycine auxotrophy was attributed to the increase in NADH concentration, which might inhibit the first step in serine/glycine biosynthesis (
14). The increase in NADH concentration was also responsible for the high resistance to INH and ETH by competitively inhibiting the binding of the INH-NAD or ETH-NAD adduct to the NADH-dependent enoyl-ACP reductase InhA (
15).
ndh mutants in both slow-growing (
M. bovis BCG) and fast-growing (
M. smegmatis) mycobacteria had no growth defect at permissive temperature, although they lost up to 90% of their ability to oxidize NADH. Interestingly, in
M. smegmatis and
M. bovis, the levels of NAD
+ cofactor stayed relatively constant despite overexpression of
ndh or mutations in
ndh, which highly reduced their NADH oxidation capability (
15), suggesting that the NAD
+ pool is tightly regulated in mycobacteria to maintain essential biochemical functions.
NDH-2 has not been reported in mammalian mitochondria, leading to the proposal that these enzymes may represent a potential drug target for the treatment of human pathogens (
6,
12,
27,
30,
31) and intracellular parasites (
32). Despite the potential of NDH-2 as a drug target, no potent nanomolar inhibitors of NDH-2 are known. Several classes of compounds are proposed to target the enzyme at micromolar concentrations (e.g., phenothiazine analogues, platanetin, quinolinyl pyrimidines) (
12,
27,
33), but the mechanism of inhibition remains unknown. Despite poor
in vitro activity, drugs of the phenothiazine family (trifluoroperazine, chlorpromazine) have potent activity
in vitro against drug-susceptible and drug-resistant
M. tuberculosis strains (
34,
35). A phenothiazine analog was also tested in a mouse model of acute
M. tuberculosis infection and was found to reduce by 90% the
M. tuberculosis bacterial load in the lungs after 11 days of treatment compared to a 3- to 4-log reduction in CFUs with the INH or rifampin control (
12). From a library of microbial products, two new compounds, scopafungin and gramicidin S, were identified as inhibitors of
M. smegmatis NDH-2, with IC
50 values better than trifluoperazine (
36). There is a crucial need for new drug targets to inhibit
M. tuberculosis, and the electron transport chain is a very attractive avenue. However, reduction in NDH-2 activity has been linked to INH and ETH resistance in both slow- and fast-growing mycobacteria (
15), and phenothiazines have been shown to be antagonistic with INH (
30). Therefore, the development of NDH-2 inhibitors will have to ascertain that interference with current drug therapy does not occur.
Some interesting questions arise from these observations. Why do mycobacteria use type II NADH dehydrogenases to recycle NADH, when they could continue to use the energy-conserving and PMF-generating NDH-I? One potential explanation is that because type II NADH dehydrogenases are non-proton-translocating, they will not be impeded by a high PMF, which could ultimately slow down metabolic flux due to back-pressure on the system. This mechanism is akin to a “relief valve” that would allow for a higher metabolic flux and ultimately higher rates of ATP synthesis, at the expense of low energetic efficiency of the respiratory chain. Second, why is
ndh an essential gene when mycobacteria could also use
nuo? The fact that
ndh is essential implies that mycobacteria do not have another mechanism to recycle NADH during normal aerobic growth. Alternatively, this is the only NADH dehydrogenase that operates under these growth conditions, and the activity of this enzyme is essential for maintaining an energized membrane. Compounds that target NDH-2 are bactericidal toward hypoxic nonreplicating
M. tuberculosis, suggesting that the respiratory chain is essential for the recycling of NADH under these conditions (
6).
Multiple Succinate Dehydrogenases and Fumarate Reductase: An Essential Link Between Central Metabolism and Respiration in Mycobacteria
Succinate dehydrogenase forms complex II of the respiratory chain and couples oxidative phosphorylation to central carbon metabolism by being an integral part of the tricarboxylic acid (TCA) cycle (
Fig. 2). This enzyme catalyzes the oxidation of succinate to fumarate wherein two electrons are transferred to quinol. The reverse reaction can be catalyzed by fumarate reductase (FRD), which is involved in anaerobic respiration (
Fig. 3). FRD and succinate dehydrogenase are closely related enzymes, and the reaction catalyzed cannot be predicted based on the primary sequence alone. Most mycobacteria harbor two annotated succinate dehydrogenases, SDH1 and SDH2 (
Table 1). SDH2 has high homology to SDH enzymes from other species and is encoded by four genes,
sdhC,
sdhD,
sdhA, and
sdhB. The genes
sdhA and
sdhB encode for the cytoplasmic part of the enzyme where the succinate to fumarate reaction takes place (SdhA), and electrons are shuttled via three iron-sulfur clusters (SdhB) to the membrane subunits SdhC and SdhD, which catalyze the electron transfer to menaquinone. The SdhA (encoded by
Rv0248c) and SdhB (
Rv0247c) subunits of SDH1 are similar to the common SDH enzyme. However, the other two genes (
Rv0250c and
Rv0249c) in the operon show no homology to membrane-bound components of SDH and FRD and are specific to the phylum
Actinobacteria. Gene expression data show that all four genes of SDH1 in
M. smegmatis are expressed in concert (
16). As an exception among mycobacteria,
M. tuberculosis encodes a third complex (
frdABCD) that is annotated as an FRD (
Table 1) and is absent in all other pathogenic strains such as
Mycobacterium avium paratuberculosis,
Mycobacterium marinum,
Mycobacterium ulcerans, and
M. leprae. Succinate dehydrogenase activity has been measured in
M. tuberculosis as well as many other mycobacterial species (
37,
38), but it is still unclear which of the three enzymes, or all, are responsible for this activity.
Mycobacteria utilize menaquinone/menaquinol (MQ/MQH
2) as a conduit between electron-donating and -accepting reactions (
Fig. 1). Menaquinone has a lower midpoint redox potential (
Em = −74 mV) compared to ubiquinone (
Em = +113 mV) and is ideally poised to donate electrons to fumarate during anaerobic conditions (
39). This means that the SDH reaction in mycobacteria (succinate oxidation to fumarate) should have an unfavorable free energy due to reverse electron flow and proton uptake to drive this reaction. It is tempting to propose that the unusual subunits of SDH1 could be the result of structural specializations in the transmembrane region to facilitate reverse electron flow from succinate to menaquinone. In fact, SDH1 and SDH2 have been shown to be differentially expressed under energy- or oxygen-limiting conditions in
M. smegmatis (
16). Under energy-limiting conditions SDH1 was upregulated 4-fold, while SDH2 was downregulated 3-fold. Under oxygen-limiting conditions, SDH1 was downregulated 30-fold, while SDH2 was upregulated 2-fold. This indicates that SDH1 could be the dedicated succinate dehydrogenase, and SDH2 catalyzes FRD activity, which is important for survival under hypoxia. This hypothesis is supported by several reports in the recent literature. A transposon-site hybridization (TraSH) screen in
M. tuberculosis suggested that SDH1 but not SDH2 was essential for growth under aerobic conditions on standard medium (
26). In contrast, in a TraSH screen selecting for mutants that continue to replicate under hypoxic conditions, Baek and coworkers (
40) found SDH2 mutants overrepresented. These results suggest that SDH2 has a pivotal role in the transition of
M. tuberculosis from aerobic to hypoxic conditions and supports the hypothesis of its being an FRD.
In fact, it has been proposed that fumarate may be an important endogenous electron acceptor for energy production and maintenance of redox balance (oxidation of NADH to NAD
+) in hypoxic nonreplicating mycobacteria (
6). Interestingly, the use of fumarate as an electron acceptor in
E. coli requires complex I, and expression of the
nuo operon is stimulated by the presence of fumarate (
41). This stands in direct contrast to
M. tuberculosis, where the
nuo operon seems to be silent under anaerobic conditions (
18). In a recent study it was shown by
13C flux analysis that
M. tuberculosis grown under hypoxia metabolizes glucose through a reverse TCA cycle to generate succinate as an excreted fermentation end product (
42). However, the metabolic flux from fumarate to succinate was unchanged in an
M. tuberculosis FRD deletion mutant, suggesting that one of the remaining putative succinate dehydrogenases (most likely SDH2) could catalyze this reaction. A more recent study suggests that the glyoxylate shunt, and not the reverse TCA, is used to metabolize both glycolytic and fatty acid carbon sources in response to oxygen limitation, and this route also produces succinate as its metabolic end product (
43). The authors propose that during oxygen limitation large amounts of succinate are produced by this pathway that are used to sustain the membrane potential, ATP synthesis, and TCA cycle precursors akin to a “metabolic battery” (
43). Moreover, because of the near neutral midpoint potential of the succinate/fumarate redox couple (+30 mV), succinate is able to bridge both oxidative and fermentative metabolic schemes depending on electron acceptor availability (
43).
The role of the
frdABCD operon in the
M. tuberculosis complex is not clear. Increased expression of this operon has been shown during carbon starvation (
20), oxygen depletion (
44), and in macrophages (
19), suggesting a role for this enzyme in persistence.
Alternative Electron Donor Utilization During Starvation and Hypoxia
During carbon starvation and slow growth, mycobacteria switch to alternative electron donors (
16,
20,
45) (
Fig. 2). Proline dehydrogenase is upregulated under both energy-limiting conditions and hypoxia (
7,
16) and is increasingly being recognized as a critical amino acid in cellular bioenergetics and redox control (
46). Proline can be utilized as an electron donor as well as a carbon and nitrogen source (
Fig. 2). The degradation of proline occurs by means of two enzymes: proline dehydrogenase (PRODH) and pyrroline-5-carboxylate dehydrogenase (P5CDH). These two enzymes catalyze the oxidation of proline to glutamate with four electrons transferred to the respiratory chain (
46). In the first step FAD is reduced to FADH
2, while in the second step NAD
+ is reduced to NADH. In some bacteria, PRODH and P5CDH are monofunctional enzymes, but in the majority of bacterial species they are fused into one protein called proline utilization A flavoenzyme PutA (
47).
In mycobacteria, PRODH and P5CDH are predicted to be monofunctional enzymes (
46). The genes encoding PRODH (
putB,
Rv1188) and P5CDH (
putA,
rocA,
Rv1187) are expressed as part of an operon (
7). It has been shown that
M. smegmatis can grow on proline as the sole carbon and energy source and also that PRODH is an important electron donor, under both energy-limiting conditions and hypoxia (
7,
16). The same authors showed that proline metabolism in mycobacteria is regulated by a unique membrane-associated transcriptional regulator called PruC (Rv1186c), encoded upstream of
pruA. The
pruAB operon with its upstream regulator
pruC is highly conserved in mycobacteria, with the exception of
M. leprae. Recent proteomics data on
M. avium paratuberculosis show that protein levels of RocA (PutA) are elevated in the intestinal tissues of cows (
48). The authors propose that
M. avium paratuberculosis has adapted to utilize proline as a carbon and nitrogen source due to the high abundance of this amino acid in the plant material that is eaten by such ruminants. Mycobacteria also encode pyrroline-5-carboxylate reductase (encoded by
proC) that catalyzes the reverse reaction of
putA, converting pyrroline-5-carboxylate to proline. Interestingly, a
proC mutant of
M. tuberculosis was avirulent in immunocompetent mice, but this was not due to its inability to proliferate intracellulary because bacterial loads increased in the mouse lungs after 20 days postinfection (
49).
Hydrogenases catalyze the reversible oxidation of molecular hydrogen: 2H
+ + 2e
− → H
2 and play a central role in energy metabolism of bacteria, archaea, and eukarya (
50). Under physiological conditions, hydrogenases couple H
2 oxidation to respiration (Knallgas reaction) or reduce protons as a way to dispose of surplus reducing equivalents. Four different types of hydrogenases are found in mycobacteria and are annotated to be of the NiFe type.
M. smegmatis harbors three (designated Hyd1, 2, and 3) of the four hydrogenase complexes (
16). Hyd1 aligns closely with group 2a uptake hydrogenases of the cyanobacteria such as
Nostoc, indicating that it oxidizes H
2 (
51). In contrast, Hyd3 is closely related to the group 3 cytoplasmic bidirectional hydrogenases. Hyd2 appears to be a founding member of the group 5 high-affinity hydrogenases (
52,
53). Hyd1, Hyd2, and Hyd3 are all soluble hydrogenases and are found in mycobacteria of the slow-growing and fast-growing type, as well as pathogenic and nonpathogenic mycobacteria (
16). However, the fourth putative hydrogenase is only found in pathogenic mycobacteria (including
M. tuberculosis complex) and seems to be restricted to slow growers (
Table 1). It shows homology to group 4 membrane-bound H
2 evolving hydrogenases. It has been shown that
M. smegmatis, among other mycobacterial species, can oxidize molecular hydrogen in the presence of carbon monoxide (CO), implying that
M. smegmatis expresses a functional hydrogenase (
54). To date, no studies have reported on the ability of mycobacteria to produce hydrogen. Gene expression studies suggest that Hyd1 and Hyd2 are used during nutrient starvation as an alternative electron source (
Fig. 2), while Hyd3 and the membrane-bound hydrogenase are more likely to have a function in disposing of electrons under anaerobic conditions (
16) (
Fig. 3). A knockout mutant of Hyd2 in
M. smegmatis showed reduced biomass production when grown on complex medium under atmospheric conditions (
16), and its homolog in
Streptomyces sp. was shown to facilitate hydrogen oxidation (
53). These data suggest that Hyd2 oxidizes hydrogen at very low concentrations, which fits with its purported role as a high-affinity hydrogenase (
52).
Carbon monoxide dehydrogenase (CO-DH) is responsible for the oxidation of CO to carbon dioxide (CO
2) in carboxydobacteria, which grow on CO as a sole source of carbon and energy (
55). Carboxydobacteria catalyze the oxidation of CO to CO
2 by the following reaction: CO + H
2O → CO
2 + 2H
+ + 2e
−. Several pathogenic and nonpathogenic mycobacteria including
M. tuberculosis are known to possess CO-DH genes. It has been shown that
M. tuberculosis H37Ra, which possesses CO-DH activity, can grow on CO as a sole source of carbon and fuel for energy generation (
56) (
Fig. 2).
Glycerol-3-phosphate dehydrogenase catalyzes the oxidation of glycerol-3-phosphate to dihydroxy-acetone phosphate and reduces either quinone or NADP (
57). In
E. coli, glycerol-3-phosphate is used either as a precursor in the biosynthesis of phospholipids or as a carbon source for energy supply (
58).
M. tuberculosis possesses genes for four predicted glycerol-3-phosphate dehydrogenases, but their role and function in mycobacterial respiration remain unknown (
Table 1).
The membrane-associated malate quinone oxidoreductase (MQO) oxidizes malate to oxaloacetate and transfers the reducing equivalents to menaquinone (
59,
60) (
Fig. 3).
M. tuberculosis harbors a copy of MQO and a cytoplasmic NAD
+-dependent malate dehydrogenase (MDH) (
61,
62). The function and role of MQO in mycobacterial respiration is unknown.
M. smegmatis lacks an MDH homolog, and it was shown that MQO responded to low oxygen concentration with a 4-fold increase in gene expression (
16). Mutants of
Corynebacterium glutamicum defective in NDH-2 activity are able to grow despite the loss of all membrane-bound NADH dehydrogenase activity (
59). The authors propose a reaction scheme whereby electron transfer from NADH to menaquinone is catalyzed by the sequential action of MDH and MQO (membrane-bound). MDH can reduce oxaloacetate with NADH to malate, and then malate is reoxidized to oxaloacetate by MQO using menaquinone as an electron acceptor (
59,
60). Support for this model comes from the observation that a Δ
mqo/ndh double mutant failed to grow under conditions where the Δ
ndh mutant grew. Furthermore,
M. smegmatis ndh mutants could be complemented by
M. bovis BCG
mdh, suggesting that such a reaction scheme might operate in mycobacteria (
15).