Mechanistic insights into protein-mediated Zinquin fluorescence

Zinquin (ZQ) is a small-molecule fluorescent probe used to monitor labile zinc in biological systems based on green fluorescence emission arising from the formation of [Zn(ZQ) 2 ] 2– complexes. However, studies in cells and protein-rich environments frequently report a pronounced blue-shifted emission, suggesting alternative interaction modes that complicate the interpretation of zinc imaging experiments. The nature of the interactions driving the blue shift is not fully understood. Here, we use the metallo-β-lactamase NDM-1 as a chemically and structurally well-defined model to elucidate the molecular origins of ZQ fluorescence in a zinc-binding protein. The NDM-1 has two distinct Zn 2+ ions in its active site, enabling direct assessment of zinc speciation, enzymatic activity, and structural integrity. Through an integrated approach combining fluorescence spectroscopy, enzyme activity assays, inductively coupled plasma mass spectrometry, X-ray absorption spectroscopy, and nuclear magnetic resonance, we identify two sources of ZQ fluorescence for this protein: formation of [Zn(ZQ) 2 ] 2– by stripping the weaker bound zinc, and a zinc-mediated ternary complex formed by NDM-1, Zn 2+ , and ZQ. This 1:1:1 complex localizes at the higher affinity zinc-binding site, where ZQ interacts concurrently with protein-bound zinc and specific residues of the NDM-1 active site. These nonpolar interactions generate a blue-shifted emission that is spectroscopically distinct from free zinc–ZQ complexes. This study characterizes the ternary complex that drives a protein-mediated fluorescence mechanism for ZQ and highlights the need to consider probe–protein interactions when interpreting fluorescent signals in chemical biology studies.