Probing in vivo Mn2+speciation and oxidative stress resistance in yeast cells with electron-nuclear double resonance spectroscopy

Abstract

Manganese is an essential transition metal that, among other functions, can act independently of proteins toeitherdefend againstorpromote oxidative stress and disease. The majority of cellular manganese exists as low molecular-weight Mn2+complexes, and the balance between opposing “essential” and “toxic” roles is thought to be governed by the nature of the ligands coordinating Mn2+. Until now, it has been impossible to determine manganese speciation within intact, viable cells, but we here report that this speciation can be probed through measurements of1H and31P electron-nuclear double resonance (ENDOR) signal intensities for intracellular Mn2+. Application of this approach to yeast (Saccharomyces cerevisiae) cells, and two pairs of yeast mutants genetically engineered to enhance or suppress the accumulation of manganese or phosphates, supports an in vivo role for the orthophosphate complex of Mn2+in resistance to oxidative stress, thereby corroborating in vitro studies that demonstrated superoxide dismutase activity for this species.