Activity of the tetrapyrrole regulator CrtJ is controlled by oxidation of a redox active cysteine located in the DNA binding domain

Abstract

CrtJ from Rhodobacter capsulatus is a regulator of genes involved in the biosynthesis of haem, bacteriochlorophyll, carotenoids as well as structural proteins of the light harvesting-II complex. Fluorescence anisotropy-based DNA-binding analysis demonstrates that oxidized CrtJ exhibits ~20-fold increase in binding affinity over that of reduced CrtJ. Liquid chromatography electrospray tandem ionization mass spectrometric analysis using DAz-2, a sulfenic acid (-SOH)-specific probe, demonstrates that exposure of CrtJ to oxygen or to hydrogen peroxide leads to significant accumulation of a sulfenic acid derivative of Cys420 which is located in the helix-turn-helix (HTH) motif. In vivo labelling with 4-(3-azidopropyl)cyclohexane-1,3-dione (DAz-2) shows that Cys420 also forms a sulfenic acid modification in vivo when cells are exposed to oxygen. Moreover, a Cys420 to Ala mutation leads to a ~60-fold reduction of DNA binding activity while a Cys to Ser substitution at position 420 that mimics a cysteine sulfenic acid results in a ~4-fold increase in DNA binding activity. These results provide the first example where sulfenic acid oxidation of a cysteine in a HTH-motif leads to differential effects on gene expression. © 2012 Blackwell Publishing Ltd.