Impact of quaternary structure upon bacterial cytochrome c peroxidases: does homodimerization matter?

All known active forms of diheme bacterial cytochrome c 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 Shewanella oneidensis (So) 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 So bCcP and an activity that is only slightly diminished (k(cat)/K(m) = 23 × 10(6) 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 E(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(L).