EFRC seminar 04/24/14

24 Apr 2014

"Hydrogenases and Oxygenases as blueprints for (bio)catalytic systems"

 

Pandelia Maria-Eirini

The Pennsylvania State University, Department of Chemistry, State College, PA16802

 

Abstract:

[NiFe] hydrogenases are enzymes of major biotechnological importance and employ exotic metallocofactors to catalyze the reversible oxidation of H2. Unfortunately, the core of their catalytic strength holds their inherent vulnerability; that is the employment of redox-active transition metal ions that have the potency to sidereact with O2 and CO leading to their (ir)reversible inactivation. It is thus of general interest to unravel the underlying determinants of O2-tolerance so as to help design enzymes or model compounds that retain their function in the presence of oxygen. Though several strategies were evolutionarily developed to overcome O2-sensitivity, such as narrow gas channels, modifications in first-coordination active site ligands, the most successful approach was engineered by a subclass of Group 1 hydrogenases. Whereas such O2-tolerant enzymes were discovered ~30 years ago and were typified by their unconventional [Fe-S] cluster EPR signals, it was only until recently that we resolved the conundrum of their O2-resistance. EPR and FTIR electrochemistry together with Mössbauer showed that in the Aquifex aeolicus enzyme, O2-tolerance is crucially linked to the unprecedented redox properties of the proximal tetranuclear Fe/S center1-3. Moreover: redox potentials of the catalytic intermediates of the [NiFe] site are similar to those of O2-sensitive enzymes, selective oxidation to Ni-B (and not Ni-A) occurs at more negative redox potentials, all [Fe-S] clusters have more positive redox potentials (≥200 mV), whereas the proximal cluster has a unique [4Fe-3S] structure accommodating three redox couples! In addition, we explored and proposed a mechanism for the CO tolerance of O2-tolerant enzymes4. Overall, combination of biochemical, biophysical, spectroscopic and bioinformatic studies led to dissect the origins O2/CO-tolerance and provide blueprints for the tunability of their properties.

Specific Group 1 hydrogenases appear to overcome their limitations utilizing an oxygenase-type like mechanism, resembling the widespread utilization of metallocofactors to harness the oxidizing power of O2. One such example is the cyanobacterial enzyme Aldehyde Deformylating Oxygenase (ADO), which has recently gained attention due to its importance in the biotechnology of biofuels. ADOs are ferritin-like enzymes that use O2 as a co-substrate to convert fatty aldehydes to alkanes. Unlike, however, typical ferritins, the reactive intermediate performs an unprecedented nucleophilic attack to the carbonyl of the aldehyde, a reaction previously observed in P450s, though with different reaction outcomes. An overview of the function and reaction mechanism of ADOs that we resolved by stopped-flow transient absorption studies, freeze-quench EPR and Mössbauer will be given5. Additionally the applicability and robustness of such spectroscopic techniques to characterize (enzymatic) mechanisms will be presented.

 

References

  1. Pandelia, M. E., Bykov, D., Izsak, R., Infossi, P., Giudici-Orticoni, M. T., Bill, E., Neese, F., and Lubitz, W. (2013) Proc Natl Acad Sci U S A 110, 483-488.
  2. Pandelia, M. E., Fourmond, V., Tron-Infossi, P., Lojou, E., Bertrand, P., Leger, C., Giudici-Orticoni, M. T., and Lubitz, W. (2010) JACS 132, 6991-7004.
  3. Pandelia, M. E., Nitschke, W., Infossi, P., Giudici-Orticoni, M. T., Bill, E., and Lubitz, W. (2011), Proc Natl Acad Sci U S A 108, 6097-6102.
  4. Pandelia, M. E., Infossi, P., Giudici-Orticoni, M. T., and Lubitz, W. (2010), Biochemistry 49, 8873-8881.
  5. Pandelia, M. E., Li, N., Norgaard, H., Warui, D. M., Rajakovich, L. J., Chang, W. C., Booker, S. J., Krebs, C., and Bollinger, J. M. (2013) JACS 135(42):15801-12.

 

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