Heterologous functionality and roles of conserved cysteine motifs of the [NiFe]-hydrogenase accessory protein, HupK/HoxV

• HupK and HoxV are involved in the biosynthesis of certain membrane-bound hydrogenases.
• HupK and HoxV are functionally equivalent.
• Cys54 of HupK is indispensable for the formation of active HupSL hydrogenase.
• Cys378 of HupK has no apparent role in the biosynthesis of HupSL hydrogenase.
• Mutant of HupK having an artificial 4-cysteine profile had significantly lower activity.

Numerous auxiliary proteins participate in the complex posttranslational modification process of [NiFe]-hydrogenases. In Thiocapsa roseopersicina, the HupK protein is important for the formation of active membrane-bound hydrogenases. The HupK proteins of various origins have moderate similarity to each other and to the large subunits of [NiFe]-hydrogenases. Site directed mutagenesis experiments were performed to disclose the role of the highly conserved cysteines in HupK. Cys54 was shown to be indispensable for the proper function of HupK and recreation of a large subunit like cysteine profile had negative effect on the activity of HupSL hydrogenase. Although, the results of the mutagenesis study slightly differed from that obtained for Ralstonia eutropha HoxV, it was clearly demonstrated that HupK from T. roseopersicina and HoxV from R. eutropha can substitute each other. It was also demonstrated that HoxV could be involved in the maturation of both Hup and Hyn hydrogenases in T. roseopersicina.