Abstract 2457 Investigation of the role of divalent metals in the RquA-catalyzed synthesis of rhodoquinone

Rhodoquinone (RQ) is an essential electron carrier used in anaerobic metabolism by select bacteria, protists and animal species. The RquA protein is required for the conversion of ubiquinone (UQ) to RQ in microbes possessing the rquA gene and may be a target for anti-microbial treatments. This reaction requires S-adenosyl-L-methionine (SAM) as an amino donor and activity is enhanced by divalent metal cations in vitro. It is currently unclear whether a metal plays a structural role or is used directly in the amino-transfer reaction mechanism. This study sought to identify metal(s) bound to native RquA and probe the requirement of these metal(s) for activity. Initial work involving EDTA inhibition of RquA identified manganese (II) as the metal cation which could best rescue activity under in vitro conditions at pH 8 (in comparison to the eight other metals tested). In this study, manganese binding to RquA was investigated using electron paramagnetic resonance (EPR). Inductively coupled plasma mass spectrometry (ICP-MS) was used to quantify metals in the native protein and isothermal calorimetry (ITC) was performed to further characterize metal binding. The metal found in highest concentration in the native protein was zinc. ITC analysis did not show evidence of Mn2+ or Zn2+ binding with RquA at pH 8; however, binding affinity experiments at pH 7 are currently underway. In vitro assays performed at pH 7 showed that Zn2+ can recover RQ production at lower concentrations, and more preferably than Mn2+, following EDTA inhibition of RquA. These results suggest that zinc is the native metal used in the RquA-mediated synthesis of RQ.