The tolerance towards oxic conditions of O2-tolerant [NiFe] hydrogenases has been attributed to an unusual [4Fe-3S] cluster that lies proximal to the [NiFe] active site. Upon exposure to oxygen, this cluster converts to a superoxidised (5+) state, which is believed to secure the formation of the so-called Ni-B state that is rapidly reactivated under reducing conditions. Here, the reductive reactivation of the membrane-bound [NiFe]-hydrogenase (MBH) from Ralstonia eutropha in a native-like lipid membrane was characterised and compared to a variant that instead carries a typical [4Fe-4S] proximal cluster. Reactivation from the Ni-B state was faster in the [4Fe-4S] variant, suggesting that the reactivation rate in MBH is limited by the reduction of the superoxidised [4Fe-3S] cluster. We propose that the [4Fe-3S] cluster plays a major role in protecting MBH by blocking the reversal of electron transfer to the [NiFe] active site, which would produce damaging radical oxygen species.