Kinetic and mechanistic studies of a de novo designed dicopper protein

The type III copper center (T3Cu) plays a major role in biology, since it is able to bind and, eventually, activate molecular oxygen1. Over the years, bioinorganic chemists have tried to replicate the peculiar reactivity and spectroscopic features of T3Cu centers. Small mimetic complexes are able to host the Cu2O2 core, nevertheless they do not present catalytic activity in aqueous solution under mild condition2. In this exciting research environment, the Artificial Metallo-Enzymes Group (AMEG) has been developing the DR (Due Rame) class of artificial metalloproteins by a de novo design approach. ApoDR1 is a dimer that binds two copper ions and, as T3Cu proteins, adopts a fourhelix bundle structure, bearing three histidine residues per monomer (Figure 1). Previous studies have demonstrated that DR1 catalyzes the oxidation of 3,5-di-tert-butylcatechol (DTBC) to the corresponding o-quinone, 3,5-di-tert-butylo-benzoquinone (DTBQ), cycling between copper(I) and copper(II) under mild conditions3. Here we present a complete kinetic study of the DR1 catalyzed catechol oxidation, which allowed to determine the kinetic parameters (Km and kcat). Interestingly, the kinetic progress curves are characterized by two phases: (i) a fast burst phase, in which DTBC rapidly binds and reduce the DR1 dicupric site; (ii) a slower conversion step, in which DTBQ is formed with very low efficiency. To get insights into the catalytic pathways for catechol oxidation by DR1, the progress curves were analyzed through different kinetic models. All models gave estimated rate constants, which suggested that the decrease in the conversion rate, after an initial burst phase (Figure 2, black arrow), was due to the ratelimiting re-oxidation step of the deoxy-DR1 dicupreous center by molecular oxygen (Figure 2, red arrow), as proposed for other model compounds of Catechol Oxidases4. Similar analysis performed on the kinetic data for a less hydrophobic substrate, namely 4-tert-butylcatechol (4TBC), strongly supported the hypothesis that DR1 is also performing catalase activity (Figure 2, green arrow), consuming hydrogen peroxide. Such uncoupled reactivity may be responsible for the slow reoxidation of the deoxy-form. [1] Solomon, E. I.; Heppner, D. E.; Johnston, E. M.; Ginsbach, J. W.; Cirera, J.; Qayyum, M.; Kieber-Emmons, M. T.; Kjaergaard, C. H.; Hadt, R. G.; Tian, L., Chem. Rev. 2014, 114 (7), 3659–3853. [2] Karahalis, G. J.; Thangavel, A.; Chica, B.; Bacsa, J.; Dyer, R. B.; Scarborough, C. C., Inorg. Chem. 2016, 55 (3), 1102–1107. [3] Chino M.; Pirro F.; Leone L.; Nastri F.; Maglio O.; Pavone V.; Lombardi A., XXVI Congresso Nazionale della Società Chimica Italiana 2017. [4] Selmeczi, K.; Réglier, M.; Giorgi, M.; Speier, G., Coordination Chemistry Reviews 2003, 245 (1–2), 191–201.