Real-Time Spectroscopy for the Analysis of Gas-processing Metalloenzymes

• Applicant: Dr. Sven Stripp
• Subject Area: Biophysics
• Term: from 2019 to 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 428046689

Within the framework of a priority program, we propose using infrared spectroscopy for biophysical investigations on iron-sulfur enzymes. This will include experiments on hydrogenases and CO dehydrogenase. Specific cofactor ligands, inhibitors, and substrates render these enzymes excellent targets for IR spectroscopy and facilitate unambiguous assignments/ assessments. In the past, I designed, established, and optimized a unique approach to real-time IR spectroscopy on water-soluble proteins, namely in situ attenuated total reflection Fourier-transform infrared spectroscopy (ATR FTIR). This technique facilitates both qualitative and quantitative spectroscopy as a function of gas supply or electrochemical potential, notably under biologically relevant conditions. At the example of [FeFe]- and [NiFe]-hydrogenases, we could show that in situ ATR FTIR spectroscopy and spectro-electrochemistry are perfectly suitable for the analysis of gas-processing metalloenzymes. Here, we propose the investigation of three interrelated topics in hydrogenases: i) the catalytic mechanism (particularly in [Fe]-hydrogenases); ii) the reaction with O2 (aerobic deactivation/inhibition);iii) the molecular proceedings of proton transfer (particularly in [FeFe]-hydrogenases). Furthermore, we are interested to understand cofactor biosynthesis. I am significantly experienced with [FeFe]- and [NiFe]-hydrogenases and will be able to expand this knowledge towards the analysis of [Fe]-hydrogenases and Ni-dependent CO dehydrogenases. Cooperation partners include renowned experts. Together with Dr. Gustav Berggren, we will introduce EPR and in situ ATR FTIR spectroscopy on whole cells to address the question whether enzymes (i.e. [FeFe]-hydrogenases) behave different in vivo and in vitro.

• DFG Programme: Priority Programmes
• Subproject of: SPP 1927: Iron-Sulfur for Life