At the cellular level, respiration requires efficient electron transfer through a series of metalloproteins that make up the Electron Transport Chain (ETC). Inefficiency in electron transfer can lead to formation of Reactive Oxygen Species (ROS). ROS can in turn produce reactive lipid-derived molecules that are known to form adducts with residues found in the proteins in the ETC. In general, such reactive residues can include histidine, but the reactivity of histidine residues that bind metals in the active site of metalloproteins has not received much consideration. To study the possibility of chemical modification at active sites, we are using diethyl pyrocarbonate (DEPC) to modify metal-ligating histidines of two key metalloproteins found in the ETC: the dicopper CuA protein from Complex IV and the [2Fe-2S] Rieske protein from Complex III. For CuA, there is one solvent-exposed active-site histidine, and cyclic voltammetry reveals a marked increase in the protein’s reduction potential upon reaction with DEPC. For the Rieske protein, two active-site histidine residues are solvent exposed, and at low DEPC concentrations voltammetric signals are observed for both one and two DEPC-histidine adducts per active site. Under basic conditions, the active-site histidine ligands are deprotonated and hence readily reactive through exposed imidazolate nitrogen atoms. However, we are also able to observe reactivity under neutral and even weakly acidic conditions. The presence of histidine reactivity at pH values well below the pKa of the metal-bound histidine indicates that proton lability must be significant for these residues. This in turn suggests that these active site residues can undergo transient proton exchange, and thus are available to assist in proton-coupled electron transfer, or even the transport of protons across a cellular (or mitochondrial) membrane during cellular respiration.

Chemical modification of metal-ligating histidines in ETC proteins

2019 Spring National Meeting

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