Nonstandard abbreviationsHyd-1, hydrogenase-1; Hyd-2, hydrogenase-2; Hyd-5, hydrogenase-5; MB, methylene blue; MBH, membrane bound hydrogenase(s); Rh. leguminosarum, Rhizobium leguminosarum; R. eutropha, Ralstonia eutrophaThe final publication is available at Springer via http://dx.doi.org/10.1007/s00775-016-1385- As well as structural genes, the Hyd-5 operon also contains several accessory genes that are predicted to be involved in different stages of biosynthesis of the enzyme. In this work, deletions in the hydF, hydG and hydH genes have been constructed. The hydF gene encodes a protein related to Ralstonia eutropha HoxO, which is known to interact with the small subunit of a[NiFe]-hydrogenase. HydG is predicted to be a fusion of the R. eutropha HoxQ and HoxR proteins, both of which have been implicated in the biosynthesis of an O2-tolerant hydrogenase, and HydH is a homologue of R. eutropha HoxV, which is a scaffold for [NiFe] cofactor assembly. It is shown here that HydG and HydH play essential roles in Hyd-5 biosynthesis. Hyd-5 can be isolated and characterised from a hydF strain, indicating that HydF may not play the same vital role as the orthologous HoxO. This study therefore emphasises differences that can be observed when comparing the function of hydrogenase maturases in different biological systems.