Insights into the molecular mechanisms of protein platination from a case study: The reaction of anticancer platinum(II) iminoethers with horse heart cytochrome C

The interactions of anticancer metallodrugs with proteins are attracting a growing interest in the current literature because of their relevant pharmacological and toxicological consequences. To understand in more depth the nature of those interactions, we have investigated the reactions of four anticancer platinum(II) iminoether complexes, namely, trans- and cis-EE (trans- and cis-[PtCl₂{(E)-HN= C(OCH₃)CH₃} ₂], respectively) and trans- and cis-Z (trans- and cis-[PtCl ₂(NH₃){(Z)-HN=C(OCH₃)CH₃}], respectively), with horse heart cytochrome c (cyt c). Our investigation was performed using mainly electrospray ionization mass spectrometry (ESI MS) but was also supported by NMR, inductively coupled plasma optical emission spectroscopy (ICP OES), and absorption electronic spectroscopy. ESI MS spectra clearly revealed the formation of a variety of platinum-protein adducts predominantly corresponding to monoplatinated cyt c species. From a careful analysis of the major ESI MS peaks, specific information on the nature of the protein-bound metallic fragments and on the underlying metallodrug-cyt c reactions was gained for the various cases. We found that trans-EE produces a major cyt c adduct (12 667 Da) that is different from that produced by either cis-EE or by trans-Z and cis-Z (12 626 Da). In particular, occurrence of extensive hydrolysis/aminolysis (the latter fostered by ammonium carbonate buffer) of the iminoether ligands and formation of the corresponding amides/amidines has been unambiguously documented. The reactivity of the iminoether ligands is greatly enhanced by the presence of cyt c as inferred from comparative NMR solution studies. Additional ESI MS measurements recorded on enzymatically cleaved samples of platinated cyt c adducts, together with NMR investigation of the cyt c/trans-EE adduct, strongly suggest that protein platination primarily occurs at Met 65. The biological and pharmacological implications of the described protein platination processes are discussed.