Structure and function of membrane proteins
Our group is interested in the structure and function of membrane proteins such as the ATP synthase, ferredoxin-NAD:oxidoreductase (Rnf), energy-conserving hydrogenase (Ech) and others. These specimens stem from unusual organisms or pathways that have evolved very early in the history of life. Therefore, these enzymes are ancestors of todays energy conserving enzymes such as, for example, complex I.
1. The Rnf complex of Acetobacterium woodii
Acetobacterium woodii has an ancient type of metabolism: it combines CO2 reduction with the synthesis of ATP (see: Microbial life under extreme energy limitation and the evolution of living matter). This pathway operates at the thermodynamic edge of life and is coupled to the generation of a transmembrane electrochemical Na+ gradient across the membrane that then drives ATP synthesis.
The Rnf complex is the only respiratory enzyme in A. woodii. It is composed of six subunits that catalyze reoxidation of reduced ferredoxin. The electrons are shuffled via iron-sulfur clusters and flavins to NAD+ that is reduced to NADH. Thus, NAD+ is the electron acceptor of this anaerobic respiratory chain (see figure below). This reaction is exergonic (E0’ Ferredoxin ≈ -500 mV; E0’ NAD/NADH = -320 mV) and coupled to the translocation of Na+ across the membrane. The low ΔG0’ of the reaction (-34 kJ/mol) allows for the transport of 1 Na+ / electron, but the stoichiometry still needs to be determined experimentally. The electron flow is reversible. The endergonic reduction of ferredoxin with NADH as reductant is made possible by energy input via Na+ influx into the cell. Actually, ∆µNa+-driven ferredoxin reduction is the function of Rnf in many aerobic and facultative anaerobic bacteria.
The Rnf complex is the prototype of a simple respiratory enzyme, evolved very early in the history of life, but kept during evolution and today found in many anaerobic, aerobic and facultative anaerobic bacteria. It is the evolutionary ancestor of the NADH: quinone-oxidoreductase (Nqr) found in some bacteria and is currently investigated in our laboratory with respect to the path of electron flow through the enzyme and its mechanistic coupling to Na+-transport.
Model of the Rnf complex in the acetogen A. woodii. Electrons are transferred from reduced ferredoxin to NAD+ by the Rnf complex that couples the electron flow to the export of Na+. CM, cytoplasmic membrane; Fd2-, reduced ferredoxin.
References
Hess, V., Schuchmann, K., Müller, V. (2013) The ferredoxin:NAD+ oxidoreductase (Rnf) from the acetogen Acetobaceterium woodii requires Na+ and is reversibly coupled to the membrane potential. J. Biol. Chem. 288 : 31496-31502.
Biegel, E., Müller , V. (2013) Electron transport in strict anaerobes. In: Encyclopedia of Biophysics (Gordon, R., ed.), Springer, Heidelberg, Germany.
Poehlein, A., Schmidt, S., Kaster, A.-K., Goenrich, M., Vollmers, J., Thürmer, A., Bertsch, J., Schuchmann, K., Voigt, B., Hecker, M., Daniel, R., Thauer, R.K., Gottschalk, G., Müller, V. (2012) An ancient pathway combining carbon dioxide fixation with the generation and utilization of a sodium ion gradient for ATP synthesis. PLoS ONE 7 : e33439.
Biegel, E., Schmidt, S., González, J.M., Müller, V. (2011) Biochemistry, evolution and physiological function of the Rnf complex, a novel ion-motive electron transport complex in prokaryotes. Cell. Mol. Life Sci. 68 : 613-634.
Biegel, E., Müller, V. (2010) Bacterial Na+ translocating ferredoxin:NAD-oxidoreductase. Proc. Natl. Acad. Sci. USA 107 : 18138-18142.
Biegel, E., Schmidt, S., Müller, V. (2009) Genetic, immunological and biochemical evidence for a Rnf complex in the acetogen Acetobacterium woodii. Environ. Microbiol. 11 : 1438-1443.
Schmidt, S., Biegel, E., Müller, V. (2009) The ins and outs of Na+ bioenergetics in Acetobacterium woodii. Biochim Biophys. Acta 1787 : 691-696.
Müller, V. (2009) Incredible anaerobes – more bioenergetic surprises to come. Environ. Microbiol. Reports 1 : 13-14.
Müller, V., Imkamp, F., Biegel, E., Schmidt, S., Dilling, S. (2008) Discovery of a ferredoxin:NAD+-oxidoreductase (Rnf) in Acetobacterium woodii: a novel potential coupling site in acetogens. Ann. N. Y. Acad. Sci. 1125 : 137-146.
Imkamp, F., Biegel, E., Jayamani, E., Buckel, W., Müller, V. (2007) Dissection of the caffeate respiratory chain in the acetogen Acetobacterium woodii: identification of a Rnf-type NADH dehydrogenase as potential coupling site. J. Bacteriol. 189 : 8145-8153.
2. The Na+ F1FO ATP synthase of A. woodii
The electrochemical Na+ potential established by the respiratory Rnf complex is used by a membrane-bound Na+ F1FO ATP synthase for ATP synthesis. The enzyme has two motors that are connected by two stalks. The cytoplasmic motor (F1) synthesizes ATP at the expense of energy provided by the membrane motor and transmitted via the central stalk. The membrane-bound motor is driven by influx of Na+. Thus, the ATP synthase is a biological nanomotor.
The membrane-embedded motor of the A. woodii enzyme has a rotor (c ring) that is made by multiple copies of different subunits. This is, so far, unique and not found anywhere else in biology. Most interestingly, one of the subunits does not have a Na+ binding site, giving a motor that “stumbles”. We have established a genetic system that allows us to modify the enzyme by site directed mutagenesis and are currently on the way to determine the function of individual rotor subunits and amino acids in catalysis.
Model of the Na+ F1Fo ATP synthase of A. woodii (A) and structure of the c subunits (B) and the unique FoVo hybrid motor of the Na+ F1Fo ATP synthase (side view (C), top view (D)). The rotor has 1 copy of c1 (grey) and 9 copies of c2 and c3 (red). Subunits c2 and c3 are encoded by two genes, but are identical on the amino acid level. The conserved glutamate of the Na+ binding site (B) and the Na+ (A, C, D) are shown in blue (Matthies et al., 2014).
References
Brandt, K., Müller, D.B., Hoffmann, J., Hübert, C., Brutschy, B., Deckers-Hebestreit, G., Müller, V. (2013) Functional production of the Na+ F1FO ATP synthase from Acetobacterium woodii in Escherichia coli requires the native AtpI. J. Bioenerg. Biomembr. 45 : 15-23.
Fritz, M., Klyszejko, A.L., Morgner, N., Vonck, J., Brutschy, B., Muller, D.J., Meier. T., Müller, V. (2008) An intermediate step in the evolution of ATPases: a hybrid FO-VO rotor in a bacterial Na+ F1FO ATP synthase. FEBS J. 275 : 1999-2007.
Fritz, M., Klyszejko, A.L., Morgner, N., Vonck, J., Brutschy, B., Muller, D.J., Meier. T., Müller, V. (2008) An intermediate step in the evolution of ATPases: a hybrid FO-VO rotor in a bacterial Na+ F1FO ATP synthase. FEBS J. 275 : 1999-2007.
Rahlfs, S., Aufurth, S., Müller, V. (1999) The Na+ -F1FO ATPase operon from Acetobacterium woodii. Operon structure and presence of multiple copies of atpE which encode proteolipids of 8 and 18 kDa. J. Biol. Chem. 274 : 33999-34004.
3. The Ech complex
Many bacteria and some archaea have an energy-conserving hydrogenase (Ech) as respiratory enzyme, often the only respiratory enzyme found in their metabolism. Like Rnf, the Ech uses reduced ferredoxin as electron donor but reduces protons, not NAD. The end product of this respiratory chain is molecular hydrogen (H2).
The Ech complex of the thermophilic acetogenic bacterium Thermoanaerobacter kivui has eight subunits and iron sulfur centers as electron carriers (see figure below). Electron flow from reduced ferredoxin to protons is coupled to the export of ions (H+ and may be Na+ in some species). Ech is the evolutionary precursor of complex I of bacterial and mitochondrial electron transport chains.
We are currently in the stage of proving ion transport by the Ech complex of T. kivui and to study its function.
Model of the Ech complex of Thermoanaerobacter kivui. Electrons are transferred from reduced ferredoxin to H+, forming molecular hydrogen. This electron flow is coupled to the export of H+ across the cytoplasmic membrane (CM).