P. denitrifi cans is a nonfermenting gram-negative facultative anaerobe that can obtain energy from either aerobic respiration or nitrate respiration.20,40,41 It is found primar-ily in soil and sewage sludge. This bacterium can grow heterotrophically on a wide variety of carbon sources, or autotrophically on H2 and CO2 under anaerobic conditions using nitrate as the electron acceptor. It can be iso-lated from soil by anaerobic enrichment with media containing H2 as the source of energy Fig. 5.14 Anaerobic respiratory chains in E. coli. When oxygen is absent, E. coli synthesizes any one of sev-eral membrane-bound reductase complexes depending upon the presence of the electron acceptors. Nitrate induces the synthesis of nitrate reductase and represses the synthesis of the other reductases. Menaquinone (E0′ = –74 mV) (or demethylmenaquinone, E0′ = –40 mV ) must be used to reduce some of the reductases (e.g., fumarate reductase, because it has a suffi ciently low midpoint potential). Ubiquinol (E0′ = +100 mV ) or menaquinone can reduce nitrate reductase because the E0′ of nitrate is 421 mV. Each reductase may be a complex of several proteins and prosthetic groups through which the electrons travel to the terminal electron acceptor. The transfer of electrons from the dehydrogenases to the reductases results in the establishment of a proton potential. If the dehydrogenase has site 1 activity, there can theoretically be two coupling sites, one at the dehydrogenase step and one linked to quinol oxidation at the reductase step. Abbreviations: cyt b, cytochrome b; Fe/S, nonheme iron–sulfur protein; FAD, fl avoprotein with fl avin adenine dinucleotide as the prosthetic group: Mo, molybdenum; TMANO, trimethylamine N-oxide; DMSO, dimethyl sulfoxide; MQ, menaquinone; UQ, ubiquinone.
and electrons, Na2CO3 as the source of carbon, and nitrate as the electron acceptor. Electron transport in P. denitrifi cans receives a great deal of research attention because certain fea-tures closely resemble electron transport in mitochondria.
Aerobic pathway
P. denitrifi cans differs from E. coli in that it has a bc1 complex and a cytochrome aa3 oxi-dase (cytochrome c oxioxi-dase), and in this way resembles mitochondria. In addition to the cytochrome aa3, there are two other terminal oxidases in the aerobic pathway.24,39 These are a different cytochrome c oxidase (cytochrome cbb3) and a ubiquinol oxidase (cytochrome bb3). (Cytochromes cbb3 are heme–copper oxidases found in several bacteria including Thiobacillus, Rhodobacter, Paracoccus, and
Bradyrhizobium.42 Cytochrome bb3 formerly was called cytochrome o or b0. But no heme o was detected when the hemes were extracted from membranes and analyzed by reversed-phase high performance liquid chromatography24,43,44). The cytochrome aa3 and cytochrome bb3 are proton pumps. (Whether cytochrome cbb3 pumps pro-tons apparently depends upon the assay con-ditions.39) The aerobic pathways as well as the sites of proton translocation are shown in Fig.
5.15A. Electrons traveling from NADH to oxy-gen can pass through as many as three coupling sites (NDH-1, bc1 complex, cyt aa3 or perhaps cyt cbb3) or as few as two coupling sites (NDH-1 and cyt bb3). Recall that the bc1 complex and cyt bb3 are coupling sites because they oxidize quinol, whereas the NDH-1 and cytochrome c oxidases are proton pumps. As described later, P. denitrifi cans also oxidizes methanol, and in
Fig. 5.15 A model for electron transport pathways in Paracoccus denitrifi cans. (A) Aerobic. The pathway has two branch points. One branch is at the level of ubiquinone, leading to one of two ubiquinol oxidases (i.e., the cyt bc1 complex or cyt bb3). Both these quinol oxidases are coupling sites. The bc1 complex extrudes two protons per electron via the Q cycle. Whereas cyt bb3 is a proton pump and extrudes one proton per electron vectorially, the second proton is extruded via a Q loop. A second branch point occurs at the level of the bc1 complex. Electrons can fl ow either to cyt aa3, which is a proton pump, or to cyt cbb3, which has been reported to pump protons under certain experimental conditions. NDH-1 is also a coupling site. (B) Anaerobic. When the bacteria are grown anaerobically, using nitrate as the electron acceptor, the cytochrome aa3 levels are very low and the electrons travel from ubiquinone to nitrate reductase and also through the bc1 complex to nitrite reductase, nitric oxide reductase, and nitrous oxide reductase.
of the membrane and reduces nitrate on the cytoplasmic side. This would create a ∆Ψ as two electrons from UQH2 fl ow electrogenically across the membrane, leaving two protons on the outside. In the cytoplasm, protons are taken up during nitrate reduction according to the fol-lowing reaction:
2H+ + 2e− + NO−3→ NO−2 + H2O
The result is the net translocation of protons to the outside, although only electrons moved across the membrane, not protons. This is the same as the Q loop described in Section 5.6.1 except that the terminal electron acceptor is nitrate instead of oxygen. The electrons to the other reductases fl ow from UQH2 through the bc1 complex, and a ∆p is generated via the Q cycle catalyzed by the bc1 complex which, as described in Section 5.6.1, is a modifi cation of the Q loop that results in the translocation of two protons per electron rather than one.
In P. denitrifi cans, Pseudomonas aeruginosa, and many other facultative anaerobes, both the synthesis and the activity of the denitrifying enzymes are prevented by oxygen. However, in certain other facultative anaerobes, includ-ing Comamonas spp., certain species of Pseudomonas, Thiosphaera pantotropha, and Alcaligenes faecalis, denitrifying enzymes are made and are active in the presence of oxy-gen.47 In these systems, both oxygen and nitrate are used simultaneously as electron acceptors, although aeration can signifi cantly decrease the rate of nitrate reduction. The advantage of co-respiration using both oxygen and nitrate is not obvious.
Periplasmic oxidation of methanol
Many gram-negative bacteria oxidize sub-stances in the periplasm and transfer the elec-trons to membrane-bound electron carriers, often via periplasmic cytochromes c. (See Section 1.2.4 for a description of the periplasm.) An example is P. denitrifi cans, which can grow aerobically on methanol (CH3OH) by oxidiz-ing it to formaldehyde (HCHO) and 2H+ with a periplasmic dehydrogenase, methanol dehy-drogenase (Fig. 5.16):
CH3OH → HCHO + 2H+
(Growth on methanol is autotrophic, since the formaldehyde is eventually oxidized to CO2, this case the electrons enter at a cytochrome c,
thus bypassing the bc1 site.
Anaerobic pathway
P. denitrifi cans can also grow anaerobically by using nitrate as an electron acceptor, reducing it to nitrogen gas in a process called denitrifi ca-tion (Fig. 5.15B).36,45 During anaerobic growth on nitrate, P. denitrifi cans has a complete citric acid cycle in which electrons are donated to the electron transport chain; but the electron trans-port chain is very different from that of aero-bic growth. The cells contain cytochrome bb3, nitrate reductase, nitrite reductase, nitric oxide reductase, and nitrous oxide reductase. (For a more complete description of these enzymes, see note 46.) The levels of cytochrome aa3 are very low. The cytochrome c shown in Fig. 5.15 is periplasmic, although there is also cytochrome c in the membrane associated with some of the electron carriers. The nitrate (NO3−) is reduced to nitrite (NO2−) in a two-electron transfer via a membrane-bound nitrate reductase.
The NO2−is reduced to nitric oxide (NO) in a one-electron transfer via a periplasmic nitrite reductase. The NO is reduced to a half-mole of nitrous acid (1/2N2O) in a one-electron step via a membrane-bound nitric oxide reductase.
And the 1/2N2O is reduced to a half-mole of dinitrogen (1/2N2) in a one-electron step by a periplasmic nitrous oxide reductase. Thus to reduce one mole of NO3− to 1/2N2 a total of fi ve electrons fl ow in and out of the cell membrane through membranous electron and periplasmic electron carriers from ubiquinol to the various reductases.
As shown in Fig. 5.15, the electron trans-port pathway includes several branches to the individual reductases. The fi rst branch site is at UQ, where electrons can fl ow either to nitrate reductase or to the bc1 complex. Then there are three branches to the three other reductases after the bc1 complex at the level of cytochrome c. In agreement with the model, electron fl ow to nitrate reductase is not sensitive to inhibitors of the bc1 complex, whereas electron fl ow to the other reductases is sensitive to the inhibitors.
The nitrate reductase spans the membrane, and it has been proposed that it creates a ∆p via a Q loop, similar to that described in Section 5.6.1.
It is proposed that the nitrate reductase accepts electrons from UQH2 on the periplasmic side