The lack of rhodanese RhdA affects the sensitivity of Azotobacter vinelandii to oxidative events

Angelo Cereda , Aristodemo Carpen , Gianluca Picariello , Gabriella Tedeschi , Silvia Pagani (1)

1 Dipartimento di Scienze Molecolari Agroalimentari

The rhdA gene of Azotobacter vinelandii codes for a rhodanese-domain protein (RhdA) with an active-site loop structure which has not currently been found in proteins of the rhodanese-homology superfamily. Considering the lack of information on the functional role of the ubiquitous rhodaneses, we undertook a study of in vivo functions of RhdA by using an A.vinelandii mutant strain (MV474) in which the rhdA gene was disrupted by deletion. Preliminary phenotypic characterization of the rhdA mutant suggested that RhdA could exert protection of Fe-S enzymes which are easy targets for oxidative damage. To highlight the role of RhdA in preserving sensitive Fe-S clusters, in the current study we analyzed the defects of the RhdA null strain by exploiting growth conditions able to enhance the catalytic deficiency of enzymes with vulnerable Fe-S clusters. We found that RhdA lack impaired A.vinelandii growth in the presence of gluconate, a carbon source that activates the Entner-Doudoroff pathway in which the first enzyme 6-phosphogluconate dehydratase employs a 4Fe-4S cluster as active-site catalyst. By combining proteomics, enzymatic profiles, and model systems to generate oxidative stress, evidence is provided that to rescue the effects of RhdA lack A.vinelandii needed to activate defensive activities against oxidative damage.The possible functionality of RhdA as a redox switch which helps A.vinelandii in maintaining cellular redox balance was investigated by using an in vitro model system that proved reversible chemical modifications in the highly reactive RhdA Cys230 thiol.

Mots clés: Life Sciences

Angelo Cereda, Aristodemo Carpen, Gianluca Picariello, Gabriella Tedeschi, Silvia Pagani. The lack of rhodanese RhdA affects the sensitivity of Azotobacter vinelandii to oxidative events. Biochemical Journal, 2009, 418 (1), pp.135-143. ⟨10.1042/BJ20081218⟩. ⟨hal-00479048⟩