Simultaneously elevating loading and activity of single atoms (SAs) is desirable for SA-containing catalysts, including single-atom catalysts (SACs). However, the fast self-nucleation of SAs limits the loading, and the activity is confined by the adsorption-energy scaling relationships on monotonous SAs. Here, we theoretically design a novel type of SA-containing catalyst generated by two-step structural self-regulation. In the thermodynamic self-regulation step, divacancies in graphene spontaneously pull up SAs from transition metal supports (dv-g/TM; TM = fcc Co, hcp Co, Ni, Cu), leading to the expectably high loading of SAs. The subsequent kinetic self-regulation step involving an adsorbate-assisted and reversible vacancy migration dynamically alters coordination environments of SAs, helping circumvent the scaling relationships, and consequently, the as-designed dv-g/Ni can catalyze NO-to-NH3 conversion at a low limiting potential of -0.25 V vs RHE.