Cobalt tetradehydrocorrins coordinated by imidazolate-like histidine in the heme pocket of horseradish peroxidase

Abstract

Horseradish peroxidase was reconstituted with cobalt tetradehydrocorrin, rHRP(Co(TDHC)), as a structural analog of cobalamin coordinated with an imidazolate-like His residue, which is generally seen in native enzymes. In contrast to the previously reported cobalt tetradehydrocorrin-reconstituted myoglobin, rMb(Co(TDHC)), the HRP matrix was expected to provide strong axial ligation by His170 which has imidazolate character. rHRP(Co^II(TDHC)) was characterized by EPR and its reaction with reductants indicates a negative shift of its redox potential compared to rMb(Co(TDHC)). Furthermore, aqua- and CN-forms of Co(III) state were prepared. The former species was obtained by oxidation of rHRP(Co^II(TDHC)) with K₃[Fe(CN)₆]. The cyanide-coordinated Co(III) species in the latter was prepared by ligand exchange of rHRP(Co^III(OH)(TDHC)) with exogenous cyanide upon addition of KCN. The ¹³C NMR chemical shift of cyanide in rHRP(Co^III(CN)(TDHC)) was determined to be 121.8 ppm. IR measurements show that the cyanide of rHRP(Co^III(CN)(TDHC)) has a stretching frequency peak at 2144 cm⁻¹. The ¹³C NMR and IR measurements indicate strong coordination of cyanide to Co^III(TDHC) relative to rMb(Co^III(CN)(TDHC)). Thus, the extent of π-back donation from the cobalt ion to the cyanide ion is relatively high in rHRP(Co^III(CN)(TDHC)). The pK _1/2 values of rHRP(Co^III(OH)(TDHC)) and rHRP(Co^III(CN)(TDHC)) are the same (pK _1/2 = 3.2) as determined by a pH titration experiment, indicating that cyanide ligation does not affect Co–His ligation, whereas cyanide ligation weakens the Co–His ligation in rMb(Co^III(CN)(TDHC)). Taken together, these results indicate that HRP reconstituted with cobalt tetradehydrocorrin is a suitable cobalamin-dependent enzyme model with imidazolate-like His residue.