The properties of a cpYFP [circularly permuted YFP (yellow fluorescent protein)] reported to act as a superoxide sensor have been re-examined in Arabidopsis mitochondria. We have found that the probe has high pH sensitivity and that dynamics in the cpYFP signal disappeared when the matrix pH was clamped by nigericin. In contrast, genetic and pharmacological manipulation of matrix superoxide had no detectable effect on the cpYFP signal. These findings question the existence of superoxide flashes in mitochondria.

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