Cj1386, an Atypical Hemin-Binding Protein, Mediates Hemin Trafficking to KatA in Campylobacter jejuni

Abstract

ABSTRACT Catalase enzymes detoxify H 2 O 2 by the dismutation of H 2 O 2 into O 2 and H 2 O through the use of hemin cofactors. While the structure and biochemical properties of catalase enzymes have been well characterized over many decades of research, it remained unclear how catalases acquire hemin. We have previously reported that Cj1386 is essential for ensuring proper hemin content in Campylobacter jejuni catalase (KatA) (A. Flint, Y. Q. Sun, and A. Stintzi, J Bacteriol 194: 334–345, 2012). In this report, an in-depth molecular characterization of Cj1386 was performed to elucidate the mechanistic details of this association. Coimmunoprecipitation assays revealed that KatA-Cj1386 transiently interact in vivo , and UV-visible spectroscopy demonstrated that purified Cj1386 protein binds hemin. Furthermore, hemin titration experiments determined that hemin binds to Cj1386 in a 1:1 ratio with hexacoordinate hemin binding. Mutagenesis of potential hemin-coordinating residues in Cj1386 showed that tyrosine 57 was essential for hemin coordination when Cj1386 was overexpressed in Escherichia coli . The importance of tyrosine 57 in hemin trafficking in vivo was confirmed by introducing the cj1386 Y57A allele into a C. jejuni Δ cj1386 mutant background. The cj1386 Y57A mutation resulted in increased sensitivity toward H 2 O 2 relative to the wild type, suggesting that KatA was not functional in this strain. In support of this finding, KatA immunoprecipitated from the Δ cj1386+cj1386 Y57A mutant had significantly reduced hemin content compared to that of the cj1386 WT background. Overall, these findings indicate that Cj1386 is involved in directly trafficking hemin to KatA and that tyrosine 57 plays a key role in this function.