The existence, and physiological relevance, of a second zinc binding (Zn2) site in MCR enzymes is a matter of continuing uncertainty. In crystal structures of the isolated catalytic domain this site, comprising His395 and His478, is completed by a coordinating Glu, from the second molecule of a crystallographic dimer . In consequence, in simulations of the isolated catalytic domain, in which this interaction is absent, the Zn2 site is unstable . In full-length MCR-1 the conserved residue Glu116, located in the MCR-1 transmembrane domain but positioned close to the catalytic center, is an alternative candidate to complete the Zn2 site. However, under all conditions tested here (presence or absence of POPE and/or PEtN addition to Thr285) the Zn2 site remained unstable unless restraints were applied to retain coordination by the His395 and His478 side chains. Thus, and consistent with the observations that as-isolated full length EptA and MCR-1 contain a single zinc equivalent, our simulations do not support the existence of a stable di-zinc form of MCR-1 (in the presence/absence of POPE) in either the resting or PEtN-modified (i.e. phosphointermediate) states. Nevertheless, our previous work supports involvement of a second zinc equivalent in PEtN transfer to the lipid A acceptor, implying that the second stage of the reaction involves recruitment of an additional zinc ion. The present data, that do not provide evidence for strong stabilization of the di-zinc form of the enzyme by the PEtN-modified phosphointermediate, then indicate that this recruitment event will likely take place at a later stage in the reaction, potentially during lipid A binding.