| Motivation Electron transfer reactions play a central role in the metabolism and bioenergetics of all organisms, yet many fundamental questions remain unsolved. Protein film voltammetry, i.e., electrochemistry of surface-confined redox enzymes, in particular when combined with surface spectroscopy and scanning-probe microscopy, is a versatile and important method to unravel the catalytic mechanism of redox enzymes. Immobilizing the protein on the electrode surface facilitates fast electron transfer, redox-state synchronization of the molecules, and instant dialysis of components in solution. Both the full biologically relevant potential window and the time domain (ranging from microseconds to hours) can be explored. The pre-steady state charging, as well as the steady state turnover rate can be directly measured as a current. However, the large majority of successful reports has relied on trial-and-error, spontaneous immobilization methods, and fortuitous electronic contact and enzyme stability. Studies on large enzyme complexes are mostly limited to soluble subunits or fragments. The bias towards more stable and soluble enzymes with at least one surface-accessible cofactor has thus far limited the number of enzymes that can studied by electro-enzymology. In most cases, the exact electron transfer pathway is unknown or non-native, which severely limits the interpretation of the data. A novel strategy for rational electrode surface functionalization is crucial to overcome these limitations and disclose hitherto unattainable mechanistic information on intra-molecular electron relay, redox-coupled catalytic reactions, and critical events such as proton translocation. Objective The objective of this proposal is to unravel the mechanism of redox-coupled processes in large respiratory enzyme complexes. In particular, membrane enzymes will be targeted that utilize the quinone pool as electron source or sink for the reduction/oxidation of water-soluble substrates. Current knowledge is biased to the half of the catalytic cycle in which substrate conversion takes place. However, the other half of the cycle, in which the active site is regenerated by intramolecular electron transfer and where crucial energy-conserving processes take place, remains poorly explored. The reason for this is that these processes are difficult to address in solution because only slow and indirect control of the redox processes in the enzymes can be achieved with freely diffusing components. Protein film voltammetry therefore is the method of choice to address this part of the mechanism, provided that a fast and well-defined electron transfer can be achieved. Here we aim to immobilize integral membrane enzymes on a rationally designed electrode surface that facilitates fast control and synchronization of the redox states of the cofactors. For this, the electrode surface will be modified with smart molecular wires that can directly plug into the quinone binding site. Approach Novel, bifunctional conjugated molecular wires will be synthesized, containing both an electrode anchoring functionality, and an attached quinone group that retains its natural redox properties and ability to bind to the enzymes. Immobilization of respiratory complexes on an electrode decorated with these wires will result in efficient electronic coupling, thus fast electron relay, and a well-defined and native electron transfer pathway. Auxiliary surface modifications will be used to accommodate and stabilize the enzyme in a bio-mimetic environment. This offers unprecedented possibilities to study electron transfer pathways and coupled reactions by tuning the driving forces both from the side of the quinone/quinol (the electrode potential) and from the side of the soluble substrate (e.g., substrate concentration, inhibitors, pH). To establish the general applicability of the rational wiring strategy, four enzymes are chosen that represent different types of respiratory systems: - Complex III (cytochrome bc1) from the denitrifying bacterium Paracoccus denitrificans - Ubiquinol oxidase (cytochrome bo3) from Escherichia coli - Membrane-bound alcohol dehydrogenase (quinohemoADH) from the nitrogen-fixing bacterium Gluconacetobacter diazotrophicus - Membrane-bound [NiFe] hydrogenase (HoxGKZ) from the knallgas bacterium Ralstonia eutropha These enzymes all add to the proton gradient that drives ATP synthesis, either by redox-driven proton ?pumping?, or by localizing the proton-associated redox reactions, but the fundamental questions regarding the nature and mechanism of redox-driven processes remain to be solved. Collaborations Local: Dr. H.A. Heering, Dr. A. Kros, Prof. M.T.M. Koper, and Prof. J. Lugtenburg at the Leiden Institute of Chemistry, and Prof. T.J. Aartsma (Biophysics) at the Leiden Institute of Physics. International: Prof. B. Ludwig (J.W. Goethe University, Frankfurt, Germany), Prof. M.E. Sosa-Torres (Universidad Nacional Autónoma de Mexico), Prof. P.M.H. Kroneck (University of Konstanz, Germany), and Prof. B. Friedrich (Humboldt University, Berlin, Germany). |
