Engineering and Tuning of Oxygen Reactivity in Heme Protein Maquettes

Abstract

There is a dearth of quantitative data related to reactive oxygen species (ROS) production, particularly superoxide, from protein systems. Understanding of ROS chemistry in proteins is necessary for avoiding damaging oxidative reactions involved in biological processes including aging and ischemic injury. These reactions are also necessary to control in order to develop robust artificial enzymes and photosystems for alternative energy production.Here we show multi-faceted control over the oxygen reactivity of ferrous heme containing designed protein maquettes. We demonstrate control over superoxide production through both inner and outer sphere electron transfer (ET) mechanisms. Inner sphere ET occurs through oxygen binding and both oxygen on and off rates effect detectible superoxide production rates and total yields. On rates are controlled via helical strain and core packing while off rates are controlled via water accessibility to the heme. The faster the on rate, the less superoxide produced due to more complete formation of the oxyferrous state. Stable oxyferrous states with half times on the order of seconds produce superoxide below the level of detection and biological relevancy. When the oxyferrous state cannot be formed due to lack of helical strain, outer sphere ET occurs at rapid rates on the order of NADPH oxidases. Faster rates of ET are observed for more solvent-exposed hemes. In all cases when the superoxide production rate is faster, we observe lower total yields. This effect is due to increased dismutation or peroxide when there are higher local concentrations of superoxide. The engineering principles we learned for superoxide control could be used to understand natural ROS processes as well as develop durable artificial ET protein systems.