Unraveling the substrate adsorption structure-performance relationship is pivotal for heterogeneous carbon supported metal single-atom catalysts (M₁/C SACs). However, due to the complexity of the functional groups on carbon material surface, it is still a great challenge. Herein, inspired by structure of enzymes, we used activated carbon (AC), which has adjustable surface oxygen functional groups (OFGs), supported atomically dispersed Fe-N₄ sites as heme-like catalyst. And based on a combination of scanning transmission electron microscopy (STEM), X-ray photoelectron spectroscopy (XPS), X-ray absorption spectroscopy (XAS), Mössbauer spectroscopy, Fourier transform infrared (FT-IR) characterizations, kinetics experiments and density functional theory (DFT) calculations, we revealed the effect of substrate adsorption behavior on AC support surface, that is, with the increase of carboxyl group in OFGs, the adsorbed 3,3′,5,5′-tetramethylbenzidine (TMB) molecular increased, and consequently the substrate enriched on AC surface. Such carboxyl group as well as Fe-N₄ active sites synergistically realized high-efficiency peroxidase-like activity, just like the heme. This work suggests that simultaneously constructing metal single-atom active sites and specific functional groups on carbon support surface may open an avenue for engineering metal-support synergistic catalysis in M₁/C SACs, which can further improve catalytic performance.