Iron to Cobalt Swapping in a Bioinspired Heme-Peroxidase: Structural Characterization and Functional Implications

Artificial heme enzymes offer unique opportunities to disentangle structure-function relationships and design novel biocatalysts. Mimochromes (MCs) are artificial, small-sized heme proteins able to reproduce the structural and functional features of natural heme enzymes. Here, we report the spectroscopic and structural investigation of a Mimochrome VI (MC6) analogue, Lys9DabMC6*a, for which we were previously able to isolate two distinct regioisomers. Mössbauer and EPR spectroscopy revealed distinct pH-dependent high-spin and quantum mixed-spin states in the Fe(III) complexes for both regioisomers. A detailed structural characterization was performed by NMR spectroscopy on the diamagnetic Co(III) analogues, providing high-resolution structures of the two isolated regioisomers. Both species show the intended helix-heme-helix sandwich fold but differ in interhelical orientation, axial histidine positioning, and second-sphere interactions, despite having the same peptide composition. Based on the reported electronic properties and structural features, we retrospectively attempt to elucidate the differences in substrate affinity and turnover frequency between the two regioisomers. Our results provide useful insights for the rational evolution of heme-based artificial minienzymes and highlight the minimal determinants required to achieve catalytic diversity.