Ribonucleotide reductase (RNR) has been extensively probed as a target enzyme in the search for selective antibiotics. Here we report on the mechanism of inhibition of nine compounds, serving as representative examples of three different inhibitor classes previously identified by us to efficiently inhibit RNR. The interaction between the inhibitors and Pseudomonas aeruginosa RNR was elucidated using a combination of electron paramagnetic resonance spectroscopy and thermal shift analysis. All nine inhibitors were found to efficiently quench the tyrosyl radical present in RNR, required for catalysis. Three different mechanisms of radical quenching were identified, and shown to depend on reduction potential of the assay solution and quaternary structure of the protein complex. These results form a good foundation for further development of P. aeruginosa selective antibiotics. Moreover, this study underscores the complex nature of RNR inhibition and the need for detailed spectroscopic studies to unravel the mechanism of RNR inhibitors.
Keywords: Diferric-oxo center; EPR; Inhibitors; Radicals; Ribonucleotide reductase; Thermal shift analysis.