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J. Bacteriol. doi:10.1128/JB.01572-06
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Identification of two catalases in Azotobacter vinelandii: a KatG homologue and a novel bacterial cytochrome c catalase, AvCCC

Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G2E9

^* To whom correspondence should be addressed. Email: bill.page{at}ualberta.ca.

	Abstract

Azotobacter vinelandii produces two detectable catalases during growth on minimal medium. The heat-labile catalase expressed during exponential growth phase was identified as a KatG homologue by LC/MS/MS from a mixed protein sample. The second catalase was heat-resistant, retaining substantial residual activity after 90°C treatment. The enzyme was purified by anion exchange and size exclusion chromatography and was found to exhibit strong absorption at 407 nm, which is often indicative of associated heme moieties. The purified protein was fragmented by proteinase K and identified by LC/MS/MS. Some identity was shared with the MauG/BCCP protein family but the enzyme exhibited a strong catalase activity never before observed in this family. Because two putative c-type heme sites (CXXCH) were predicted in the peptide sequence and were demonstrated experimentally, the enzyme was designated a cytochrome c catalase (CCC). However the local organization of the AvCCC heme motifs differed significantly from that of the BCCPs as the sites were confined to the C-terminal half of the catalase. A possible Ca²⁺ binding motif, previously described in the BCCPs, is also present in the AvCCC peptide sequence. Some instability in the presence of EGTA was observed. Expression of the catalase was abolished in cccA mutants resulting in a nearly 8,700-fold reduction in peroxide resistance in stationary phase.