Chemistry and the Environment, Contributed Talk (15min)

Mechanistic implications of oxygen uncoupling in two Rieske non-heme iron dioxygenases

C. E. Bopp1,3, S. G. Pati2, H. E. Kohler1,3, T. B. Hofstetter1,3*
1Eawag, Swiss Federal Institute of Aquatic Science and Technology, Department of Environmental Chemistry, Überlandstrasse 133, 8600 Dübendorf, Switzerland, 2University of Basel, Biogeochemistry Research Group, Bernoullistrasse 30, 4056 Basel, Switzerland, 3ETH Zürich, Institute of Biogeochemistry and Pollutant Dynamics, Universitätsstrasse 16, 8092 Zürich, Switzerland

Rieske non-heme iron dioxygenases (RDOs) are the key enzymes responsible for initial steps of aerobic biotransformation of numerous persistent aromatic contaminants in the environment. Knowledge of the catalytic mechanism, substrate specificity, and activity of RDOs enables one to assess which environmental contaminant can be fed into common metabolic pathways through dihydroxylation steps leading to cis-dihydrodiols and catechol-type products. However, while the strategies of O2 activation and control of reactive Fe-oxygen species of many mononuclear non-heme iron oxygenases have been studied in detail, the efficiency of substrate oxygenation is largely unknown. Current hypotheses suggest that RDO substrates other than the native ones are hydroxylated only poorly and thus give rise to so-called O2 uncoupling and concomitant formation of reactive oxygen species [1]. Given that RDO-expressing microorganisms are exposed to complex mixtures of structurally similar aromatic compounds at contaminated sites, O2 uncoupling could even be the predominant outcome of enzymatic activity.

In this study, we explored the relevance of O2 uncoupling pathways of RDOs as well as its implications for the kinetics and mechanisms of O2 activation and substrate oxygenation. We studied two closely related nitroarene dioxygenases, namely nitrobenzene dioxygenase (NBDO) and 2-nitrotoluene dioxygenase (2NTDO) with a wide range of substrates [2,3]. Substrate-specific uncoupling was observed through measurements of O2 consumption relative to product formation in purified enzyme assays and revealed an extent of O2 uncoupling of 30% to 100% of the activated O2. The efficiency of oxygenation by NDBO showed preference for meta-substituted nitroarene substrates as opposed to ortho-substituted substrates for 2NTDO. Conversely, 18O kinetic isotope effects (KIEs) used for characterization of reactive Fe-oxygen species were between 1.015 and 1.025 which is indicative of Fe(III)-peroxo species formation and lacked any substrate specificity. 13C KIEs of substrate hydroxylation correlated with the extent of O2 uncoupling of NBDO whereas these numbers were generally close to unity for 2NDTO. Our observations suggest catalytic mechanisms of RDOs in which the timing of O2 uncoupling and release of seemingly unreacted substrate are both substrate- and enzyme-specific. The high share of unproductive O2 activation challenges the widely made assumption of enzyme evolution towards an efficient oxygenation of preferred substrates.

[1] Charlotte E. Bopp, Hans Peter E. Kohler, Thomas B. Hofstetter, Chimia, 2020, 74, 108-114.
[2] Sarah G. Pati, Hans Peter E. Kohler, Anna Pabis, Piotr Paneth, Rebecca E. Parales, Thomas B. Hofstetter, Environmental Science and Technology, 2016, 50, 6708-6716.
[3] Sarah G. Pati, Hans Peter E. Kohler, T.B. Hofstetter Methods in Enzymology, 2017, 596, 291-329.