Impact of Quaternary Structure upon Bacterial Cytochrome <i>c</i> Peroxidases: Does Homodimerization Matter?

All known active forms of diheme bacterial cytochrome <i>c</i> peroxidase (bCcP) enzymes are described by a homodimeric state. Further, the majority of bCcPs reported display activity only when the high-potential electron transfer heme of the protein (Fe_H) is reduced to the ferrous oxidation state. Reduction of Fe_H results in a set of conformational changes allowing for the low-potential peroxidatic heme (Fe_L) to adopt a high-spin, five-coordinate state that is capable of binding substrate. Here we examine the impact of dimerization upon the activity of the <i>Shewanella oneidensis</i> (<i>So</i>) bCcP by the preparation of single charge-reversal mutants at the dimer interface and use the resulting constructs to illustrate why dimerization is likely a requirement for activity in bCcPs. The E258K mutant is found to form a monomeric state in solution as characterized by size exclusion chromatography and analytical ultracentrifugation analyses. The resulting E258K monomer has an unfolding stability comparable to that of wild-type <i>So</i> bCcP and an activity that is only slightly diminished (<i>k</i>_cat/<i>K</i>_m = 23 × 10⁶ M^–1 s^–1). Spectroscopic and potentiometric analyses reveal that while the thermodynamic stability of the activated form of the enzyme is unchanged (characterized by the <i>E</i>_m value of the Fe_H^II/Fe_H^III couple), the kinetic stability of the activated form of the enzyme has been greatly diminished upon generation of the monomer. Together, these data suggest a model in which dimerization of bCcP enzymes is required to stabilize the lifetime of the activated form of the enzyme against reoxidation of Fe_H and deactivation of Fe<sub>L