In the human gastric bacterium, Helicobacter pylori, two metallo-enzymes, hydrogenase and urease, are essential for in vivo colonization, the latter being a major virulence factor. The UreA and UreB structural subunits of urease, and UreG, one of the accessory proteins for Ni²⁺ incorporation into apo-urease were taken as baits for tandem affinity purification (TAP). The method allows the purification of protein complexes under native conditions and physiological expression levels of the bait protein. Furthermore, the TAP technology was combined with in vivo crosslink to capture transient interactions. The results revealed different populations of urease complexes: (i), urease captured during activation by Ni²⁺ ions, comprising all the accessory proteins, and (ii), urease in association with metabolic proteins involved e.g. in ammonium incorporation and the cytoskeleton. Using UreG as a bait protein, we copurified HypB, the accessory protein for Ni²⁺ incorporation into hydrogenase, reported to play a role in urease activation. The interactome of HypB partially overlapped with that of urease and revealed interactions with SlyD, known to be involved in hydrogenase maturation as well as with proteins implicated in the formation of [Fe-S] clusters, present in the small subunit of hydrogenase. In conclusion, this study provides new insight into coupling of ammonium production and assimilation in the gastric pathogen and the intimate link between urease and hydrogenase maturation.