Structural and Mutagenesis Studies on the Cytochrome c Peroxidase from Rhodobacter capsulatus Provide New Insights into Structure-Function Relationships of Bacterial Di-heme Peroxidases

Lina De Smet,Savvas N Savvides,Ellen Van Horen,Graham Pettigrew,Jozef J Van Beeumen

doi:10.1074/jbc.m509582200

Lina De Smet, Savvas N Savvides + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m509582200

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Abstract

Cytochrome c peroxidases (CCP) play a key role in cellular detoxification by catalyzing the reduction of hydrogen peroxide to water. The di-heme CCP from Rhodobacter capsulatus is the fastest enzyme (1060 s(-1)), when tested with its physiological cytochrome c substrate, among all di-heme CCPs characterized to date and has, therefore, been an attractive target to investigate structure-function relationships for this family of enzymes. Here, we combine for the first time structural studies with site-directed mutagenesis and spectroscopic studies of the mutant enzymes to investigate the roles of amino acid residues that have previously been suggested to be important for activity. The crystal structure of R. capsulatus at 2.7 Angstroms in the fully oxidized state confirms the overall molecular scaffold seen in other di-heme CCPs but further reveals that a segment of about 10 amino acids near the peroxide binding site is disordered in all four molecules in the asymmetric unit of the crystal. Structural and sequence comparisons with other structurally characterized CCPs suggest that flexibility in this part of the molecular scaffold is an inherent molecular property of the R. capsulatus CCP and of CCPs in general and that it correlates with the levels of activity seen in CCPs characterized, thus, far. Mutagenesis studies support the spin switch model and the roles that Met-118, Glu-117, and Trp-97 play in this model. Our results help to clarify a number of aspects of the debate on structure-function relationships in this family of bacterial CCPs and set the stage for future studies.

Highlights

The overall reaction for the bacterial di-heme peroxidases is similar to that in yeast and horseradish peroxidases, but the catalytic mechanism is significantly different
With the exception of the Nitrosomonas europaea enzyme [3], bacterial CCP (BCCP) are only active in the mixed valence state in which the high potential, electron transferring heme is in the reduced state and the peroxidatic, low potential heme is in the Fe(III) state (4 –7)
Two distinct structural snapshots of Cytochrome c peroxidases (CCP) from P. nautica became available as the result of the serendipitous reduction of the C-terminal heme group by synchrotron X-rays used for the structural studies [10]

Summary

EXPERIMENTAL PROCEDURES

Crystallization and Structure Determination—Recombinant R. capsulatus CCP was prepared and crystallized as previously described [7, 25]. The periplasmic fraction was loaded onto a Q-Sepharose column and eluted with a step gradient of 0 –500 mM NaCl. The fractions between 0.2 and 0.3 M NaCl were pooled and concentrated using ammonium sulfate precipitation. The BCCP-containing fractions were separated on an octyl-Sepharose hydrophobic interaction column. The BCCP was subjected to anion-exchange chromatography. The enzyme fractions were pure according to SDS-PAGE and mass-spectrometric analysis [7]. Crystals suitable for x-ray diffraction analysis were obtained from 25% (w/v) polyethyleneglycol monomethyl

Data collection

RESULTS AND DISCUSSION

LP heme reduced