A simple energy-conserving system: proton reduction coupled to proton translocation

Abstract

Oxidative phosphorylation involves the coupling of ATP synthesis to the proton-motive force that is generated typically by a series of membrane-bound electron transfer complexes, which ultimately reduce an exogenous terminal electron acceptor. This is not the case with Pyrococcus furiosus, an archaeon that grows optimally near 100 degrees C. It has an anaerobic respiratory system that consists of a single enzyme, a membrane-bound hydrogenase. Moreover, it does not require an added electron acceptor as the enzyme reduces protons, the simplest of acceptors, to hydrogen gas by using electrons from the cytoplasmic redox protein ferredoxin. It is demonstrated that the production of hydrogen gas by membrane vesicles of P. furiosus is directly coupled to the synthesis of ATP by means of a proton-motive force that has both electrochemical and pH components. Such a respiratory system enables rationalization in this organism of an unusual glycolytic pathway that was previously thought not to conserve energy. It is now clear that the use of ferredoxin in place of the expected NAD as the electron acceptor for glyceraldehyde 3-phosphate oxidation enables energy to be conserved by hydrogen production. In addition, this simple respiratory mechanism readily explains why the growth yields of P. furiosus are much higher than could be accounted for if ATP synthesis occurred only by substrate-level phosphorylation. The ability of microorganisms such as P. furiosus to couple hydrogen production to energy conservation has important ramifications not only in the evolution of respiratory systems but also in the origin of life itself.

Fig. 1.

Model of chemiosmotic coupling between the MBH and the ATP synthase in intact cells of P. furiosus. Fd_red and Fd_ox represent the reduced (by oxidative metabolism) and oxidized forms of the redox protein Fd, respectively. Fd_red is reoxidized by the MBH, which reduces H⁺ to produce H₂. As the primary proton-translocating complex, MBH also pumps H⁺ across the membranes, resulting in a transmembrane proton gradient. This gradient can then be used by the secondary proton translocating complex, ATPase, to produce ATP. See text for details, and note that experiments were conducted with inverted vesicles.

Fig. 2.

ATPase and hydrogen evolution activities of washed membrane vesicles of P. furiosus. Enzyme activities were measured as described in Materials and Methods. Fd was used as the electron carrier for H₂ production. ATPase activity is specifically inhibited by DCCD and nitrate but not by azide. The membrane-bound hydrogenase activity is specifically inhibited by copper ions (CuCl₂).

Fig. 3.

Hydrogen production and ATP synthesis catalyzed simultaneously by membrane vesicles. Enzyme activities were measured as described in Materials and Methods. Fd or MV was used as the electron carrier for H₂ production. Membrane vesicles synthesize ATP and produce H₂ in the presence of either Fd or MV as electron carriers (complete). MBH-coupled ATP synthesis is not observed either in the absence of the electron donor (–DT) for H₂ production or if the MBH is inhibited (+CuCl₂). ATP is not produced in the presence of ATP-synthase inhibitors (+DCCD, +nitrate) or in the absence of the pmf (+CCCP).

Fig. 4.

Direct measurements of ΔpH and Δψ during hydrogen production. The change in pH (a and b) was measured by using acridine orange, and the change in electrochemical potential (c and d) was measured by using oxonol VI as described in Materials and Methods. ΔpH (a) and Δψ (c) are formed when the electron donor Fd is added to the vesicles. Δψ and ΔpH are collapsed by the addition of CCCP (a and c) and when the MBH is inhibited by copper ions (CuCl₂, d), and these also prevent formation of ΔpH.

Fig. 5.

Hydrogen oxidation driven by reverse proton pumping. H₂ uptake activity was measured by using benzyl viologen (BV) or MV as the electron acceptor as described in Materials and Methods. The activity is stimulated by the presence of ATP (+ATP) but is unaffected by ADP and P_i [+(ADP + P_i)] or when the ATPase is inhibited by nitrate (+nitrate).

Fig. 6.

Pathways of electron flow and energy conservation during sugar catabolism by P. furiosus. G-6-P, glucose 6-phosphate; F-6-P, fructose 6-phosphate; F-1,6-bP, fructose 1,6-bisphosphate; GAPOR, GAP:Fd oxidoreductase; 3-PG, 3-phosphoglycerate; 2-PG, 2-phosphoglycerate; PEP, phosphoenolpyruvate; POR, pyruvate:Fd oxidoreductase; ACS, acetyl-CoA synthase. See text for details.