Evidence that azide occupies the chloride binding site near the manganese cluster in photosystem II

The effect of adding azide to photosystem II (PS II) membrane samples (BBY preparation), with or without chloride, has been investigated using continuous wave (CW) and pulsed EPR spectroscopy. In the BBY samples with 25 mM chloride, we observed that the inhibition induced by azide is partly recovered by the addition of bicarbonate. Electron spin-echo envelope modulation (ESEEM) was used to search for spin transitions of 15N nuclei magnetically coupled to the S2 state Mn cluster (multiline EPR signal form) in 15N (single terminal label) azide-treated samples with negative results. However, an 15N ESEEM peak was observed in parallel chloride-depleted PS II samples when the 15N-labeled azide is added. However, this peak is absent in chloride-depleted samples incubated in buffer containing both chloride and [15N]azide. Thus these results demonstrate an azide binding site in the immediate vicinity of the Mn cluster, and since this site appears to be competitive with chloride, these results provide further evidence that chloride is bound proximal to the Mn cluster as well. Discussion on the possible interplay between azide, chloride, and bicarbonate is provided.