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

ABSTRACT: 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).

SUBMITTER: Ellis KE

PROVIDER: S-EPMC3880548 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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

Ellis Katie E KE Frato Katherine E KE Elliott Sean J SJ

Biochemistry 20121205 50

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 ...[more]

PMID: 23189923

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Project description:BackgroundType III secretion systems are a common virulence mechanism in many Gram-negative bacterial pathogens. These systems use a nanomachine resembling a molecular needle and syringe to provide an energized conduit for the translocation of effector proteins from the bacterial cytoplasm to the host cell cytoplasm for the benefit of the pathogen. Prior to translocation specialized chaperones maintain proper effector protein conformation. The class II chaperone, Invasion plasmid gene (Ipg) C, stabilizes two pore forming translocator proteins. IpgC exists as a functional dimer to facilitate the mutually exclusive binding of both translocators.ResultsIn this study, we present the 3.3 A crystal structure of an amino-terminally truncated form (residues 10-155, denoted IpgC10-155) of the class II chaperone IpgC from Shigella flexneri. Our structure demonstrates an alternative quaternary arrangement to that previously described for a carboxy-terminally truncated variant of IpgC (IpgC1-151). Specifically, we observe a rotationally-symmetric "head-to- head" dimerization interface that is far more similar to that previously described for SycD from Yersinia enterocolitica than to IpgC1-151. The IpgC structure presented here displays major differences in the amino terminal region, where extended coil-like structures are seen, as opposed to the short, ordered alpha helices and asymmetric dimerization interface seen within IpgC1-151. Despite these differences, however, both modes of dimerization support chaperone activity, as judged by a copurification assay with a recombinant form of the translocator protein, IpaB.ConclusionsFrom primary to quaternary structure, these results presented here suggest that a symmetric dimerization interface is conserved across bacterial class II chaperones. In light of previous data which have described the structure and function of asymmetric dimerization, our results raise the possibility that class II chaperones may transition between asymmetric and symmetric dimers in response to changes in either biochemical modifications (e.g. proteolytic cleavage) or other biological cues. Such transitions may contribute to the broad range of protein-protein interactions and functions attributed to class II chaperones.

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Dataset Information

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

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

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