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

Abstract

The rhdA gene of Azotobacter vinelandii codes for RhdA, a rhodanese-domain protein 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, in the present study we examined the 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 over Fe-S enzymes, which are easy targets for oxidative damage. To highlight the role of RhdA in preserving sensitive Fe-S clusters, in the present study we analysed the defects of the rhdA-null strain by exploiting growth conditions which resulted in enhancing the catalytic deficiency of enzymes with vulnerable Fe-S clusters. We found that a lack of RhdA 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 an active-site catalyst. By combining proteomics, enzymatic profiles and model systems to generate oxidative stress, evidence is provided that to rescue the effects of a lack of RhdA, 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 the cellular redox balance was investigated by using an in vitro model system that demonstrated reversible chemical modifications in the highly reactive RhdA Cys(230) thiol.

The rhodanese RhdA helps Azotobacter vinelandii in maintaining cellular redox balance

“…Although the relative increases of oxidized glutathione in the mutant were comparable in both growth conditions, the levels of oxidized glutathione were higher in gluconate growths, a condition in which growth defects of MV474 have been observed (Cereda et al, 2009), and ROS production in the absence of RhdA was elicited ( Figure 1A; gray bars).…”

“…The phenotypic characterization of the A. vinelandii rhdA mutant strain MV474 (Cereda et al, 2007(Cereda et al, , 2009 clearly showed that inactivation of enzymes containing labile Fe-S clusters (Imlay, 2006) was enhanced in the absence of RhdA. Our goal was, therefore, to explore whether these effects could correlate with an altered redox balance in A. vinelandii cells lacking RhdA.…”

“…Furthermore, in conditions of provoked oxidative stress by addition of the superoxide generator phenazine methosulfate (PMS), a stressor-dependent induction of the rhdA gene provided a link between RhdA and the PMS-resistance of the wild-type strain previously observed (Cereda et al, 2007(Cereda et al, , 2009. The potentiality of RhdA in buffering the general levels of oxidants in A. vinelandii cells is discussed.…”

“…The first indication that RhdA could protect A. vinelandii from oxidative events stemmed from the observation that in the presence of the superoxide generator PMS, A. vinelandii growth was significantly impaired only in the rhdA mutant strain (Cereda et al, 2007(Cereda et al, , 2009 highlight whether the presence of RhdA could buffer the effects of oxidative stress.…”

“…Accordingly, the defects of the A. vinelandii RhdA null mutant was elicited by using growth conditions that enhance the catalytic deficiency of enzymes with vulnerable Fe-S clusters (Cereda et al, 2009). However, in the presence of gluconate as carbon source, which activates the EntnerDoudoroff pathway (Peekhaus and Conway, 1998) where the first enzyme 6-phosphogluconate dehydratase contains a vulnerable 4Fe-4S cluster (Gardner and Fridovich, 1991;Imlay, 2006), it was found that defensive activities against oxidative damage were needed to rescue the effects of the lack of RhdA in A. vinelandii MV474.…”