The heme binding protein ChuX is a regulator of heme degradation by the ChuS protein in Escherichia coli O157:H7

Escherichia coli O157:H7 possesses an 8-gene cluster (chu genes) that contains genes involved in heme transport and processing from the human host. Among the chu genes, four encode cytoplasmic proteins (ChuS, ChuX, ChuY and ChuW). ChuX was previously shown to be a heme binding protein and to assist ChuW in heme degradation under anaerobic conditions. The purpose of this work was to investigate if ChuX works in concert with ChuS, which is a protein able to degrade heme by a non-canonical mechanism and release the iron from the porphyrin under aerobic conditions using hydrogen peroxide as the oxidant. We showed that when the heme-bound ChuX and apo-ChuS protein are mixed, heme is efficiently transferred from ChuX to ChuS. Heme-bound ChuX displayed a peroxidase activity with ABTS and H2O2 but not heme-bound ChuS, which is an efficient test to determine the protein to which heme is bound in the ChuS-ChuX complex. We found that ChuX protects heme from chemical oxidation and that it has no heme degradation activity by itself. Unexpectedly, we found that ChuX inhibits heme degradation by ChuS and stops the reaction at an early intermediate. We determined using surface plasmon resonance that ChuX interacts with ChuS and that it forms a relatively stable complex. These results indicate that ChuX in addition to its heme transfer activity is a regulator of ChuS activity, a function that was not described before for any of the heme carrier protein that delivers heme to heme degradation enzymes. The ChuX protein binds heme and protects it from chemical degradation. Upon mixing with the ChuS protein, ChuX transfers its heme to ChuS. Exposure of the ChuX-ChuS complex to ascorbate or hydrogen peroxide leads to heme degradation that is arrested at an intermediate step instead of a complete reaction. [Display omitted] •ChuX binds and protects heme from chemical oxidation.•ChuX has no heme degradation activity.•ChuX readily transfers its heme to the ChuS protein.•ChuX forms a complex that inhibits the heme degradation reaction catalysed by ChuS.