Comparative Analysis of Cyanobacterial and Plant Peroxiredoxins and Their Electron Donors: Peroxidase Activity and Susceptibility to Overoxidation

Peroxiredoxins (Prxs)are peroxidases that use thiol-based catalytic mechanisms implying redox-active cysteines. The different Prx families have homologs in all photosynthetic organisms, including plants, algae, and cyanobacteria. However, recent studies show that the physiological reduction systems that provide Prxs with reducing equivalents to sustain their activities differ considerably between cyanobacterial strains. Thus, for example, the filamentous cyanobacterium Anabaena sp. PCC 7120 is similar to the chloroplast in that it possesses an abundant 2-Cys Prx, which receives electrons from the NADPH-dependent thioredoxin reductase C (NTRC). In contrast, the unicellular cyanobacterium Synechocystis sp. PCC 6803, which lacks NTRC, has little 2-Cys Prx but high amounts of PrxII and 1-Cys Prx. The characterization of cyanobacterial Prxs and their electron donors relies on straightforward enzymatic assays and tools to study the physiological relevance of these systems. Here, we present methods to measure peroxidase activities in vitro and peroxide decomposition in vivo. Several approaches to detect overoxidation of the active site cysteine in cyanobacterial 2-Cys Prxs are also described.