Fluorescence labels as sensors for oxygen binding of arthropod hemocyanins

The molecular basis of high cooperativity in multi-subunit proteins is still unknown in most cases. Oxygen binding by multi-subunit hemocyanins produces two intrinsic spectroscopic signals which are, however, either limited to the UV or are very weak. Here we demonstrate that fluorescence labels emitting in the visible can be used as sensors for cooperative oxygen binding of hemocyanins. Fluorescence resonance energy transfer to the oxygenated active sites quenches the emission of the labels by roughly 50% upon oxygenation of the protein. The labels give strong and photo-stable emission, allowing imaging of single hemocyanin molecules. Therefore, this study opens up a new perspective for investigating the molecular basis of cooperative oxygen binding at the single-molecule level. In addition, another novel application is provided by these labels, i.e., the investigation of the influence of effectors by recording simultaneously the binding of oxygen in the visible and of effectors in the UV.