Ferrochelatase catalyzes the terminal step of heme biosynthesis by inserting ferrous iron into protoporphyrin IX. The current study is aimed at understanding the structural basis of porphyrin binding and distortion in ferrochelatase-catalyzed reaction by functional analysis of a highly conserved active site loop motif. The loop was shown to contact bound porphyrin based on crystallographic and molecular modelling observations, and its role in murine ferrochelatase was assessed by random mutagenesis and steady-state kinetic analysis. To overcome the limitations of conventional kinetic assay methods for ferrochelatase, a continuous assay was developed by monitoring porphyrin fluorescence decrease using natural substrates ferrous iron and protoporphyrin IX under anaerobic conditions. For wild-type murine ferrochelatase, the assay yielded KmPPIX of 1.4 uÌ?M, KmFe2+ of 1.9 uÌ?M and kcat of 4.0 min-1 at 30 °C. The results of random mutagenesis indicated that all the loop re sidues spanning Q248-L257 tolerated functional substitutions. While Q248, S249, G252, W256 and L257 possessed high informational content, the other five positions contained low informational content. Selected active loop variants exhibited kcat comparable to or higher than that of wild-type enzyme, while KmPPIX was increased in most variants. The kcat/KmPPIX remained largely unchanged, with the exception of a 10-fold reduction in variant K250M/V251L/W256Y. Molecular modeling of the active loop variants suggested that loop mutations resulted in alterations of the active site architecture. Distortion of porphyrin substrate, a crucial step in ferrochelatase-catalyzed metal chelation, was examined using resonance Raman spectroscopy. The results revealed that both wild-type enzyme and loop variants induced saddling of substrate protoporphyrin. Further, loop mutations generally interfered with porphyrin saddling, with the least deformation observed in variant K250M/V251L/W256Y.N-alkyl porphyrins are potent competitive inhibitors of mammalian ferrochelatase. The present study showed that while N-methyl protoporphyrin strongly inhibited the wild-type enzyme with an inhibition constant in the nanomolar range, it was less effective in inhibiting variants P255R and P255G. These results suggest that inhibitor binding may be associated with a protein conformational change mediated by P255. Wild-type ferrochelatase is a homodimeric [2Fe-2S] cluster-containing protein. Variants S249A/K250Q/V251C and S249A/K250R/G252W were found to retain enzymatic activity in the absence of FeS cluster and form active, higher order oligomers. These observations raise the possibility that FeS cluster and homodimeric organization are not essential to ferrochelatase catalysis.