Sulfur Biochemistry and Microbial Bioenergetics
PD Dr. Arnulf Kletzin
The oxidation and reduction of elemental sulfur (S) and inorganic sulfur species are some of the most important energy-yielding reactions for microorganisms living in volcanic hot springs, solfataras, and submarine hydrothermal vents. In addition, bioleaching of sulfide minerals – especially copper – by thermophilic Archaea and Bacteria like Acidianus, Metallosphaera, and Sulfobacillus became a well established technology, which is widely applied in metal extraction from low-grade ores.
In our group, the biochemistry of the oxidation and reduction reactions of S as primary inorganic energy source by acidophilic microorganisms is studied. Acidianus ambivalens, our model organism, grows in volcanic hot springs and solfataras at 80˚C and pH 2.5. It utilizes S and other sulfur compounds as the sole energy source and requires a gas phase of CO2 and either air (aerobic growth) or H2 (anaerobic growth). When growing aerobically, A. ambivalens oxidizes elemental sulfur to sulfuric acid as electron donor and energy source. When growing anaerobically, it reduces sulfur with H2 as electron donor to H2S.
We purified several soluble and membrane-bound enzymes of the oxidation and reduction pathways including a soluble sulfur oxygenase reductase (SOR), a membrane-bound thiosulfate:quinone oxidoreductase, a soluble tetrathionate hydrolase, and a membrane-bound complex of hydrogenase and sulfur reductase. Our collaborators from Oeiras, Portugal, complement our efforts by elucidating large parts of the electron transport chain of this archaeon (Miguel Teixeira, Manuela M. Pereira, Cláudio Gomes, Carlos Frazão, Margarida Archer, Instituto de Tecnologia Química et Biológica; http:///www.itqb.unl.pt).
The 3D structure of the SOR was solved and it showed that the protein forms a large hollow sphere with a secluded inner cavity and mononuclear non-heme iron sites. The future goal is to elucidate the reaction mechanisms of each of the enzymes in molecular detail. With the SOR we use a combination of site-directed mutagenesis, spectroscopy, and X-ray crystallography of mutant proteins. Less is known about the other enzymes where we try to obtain 3D and kinetic data. We already solved another structure of a small thermostable Rieske ferredoxin. In the future we will extend our efforts understanding the reaction mechanisms of several of these enzymes in molecular detail. In addition we will especially try to turn the SOR into a nanoscale reaction chamber for other substrates.
It is not very well known which parts of the primary and secondary coordination spheres of a metal site within a given protein contributes to the apparent reduction potential of the metal site(s). We will use both proteins – the Rieske and the SOR – to experimentally change the coordination environment in order to fine-tune metal sites to fit predicted reduction potentials.
Surface Layer Proteins of Archaea
S-layer proteins of Acidianus and related Sulfolobus strains were studied as possible attachment mediators for the solid growth substrate sulfur. Together with Reinhard Rachel (Regensburg) and Sonja V. Albers (Groningen, NL, now: Marburg) we elucidated novel transcription patterns of the genes. We characterized the proteins structurally and generated computer-derived models, which has not been achieved previously for these proteins. In the future we will analyze the properties of these proteins as anchors for extracellular (sulfur-metabolizing) enzymes and as regular two-dimensional surface structures with biotechnological potential in conjunction with a prospected research initiative on Bio-Functionalization of Surfaces.