Giardia intestinalis Incorporates Heme into Cytosolic Cytochromeb5

Abstract

ABSTRACTThe anaerobic intestinal pathogenGiardia intestinalisdoes not possess enzymes for heme synthesis, and it also lacks the typical set of hemoproteins that are involved in mitochondrial respiration and cellular oxygen stress management. Nevertheless,G. intestinalismay require heme for the function of particular hemoproteins, such as cytochromeb5(cytb5). We have analyzed the sequences of eukaryotic cytb5proteins and identified three distinct cytb5groups: group I, which consists of C-tail membrane-anchored cytb5proteins; group II, which includes soluble cytb5proteins; and group III, which comprises the fungal cytb5proteins. The majority of eukaryotes possess both group I and II cytb5proteins, whereas threeGiardiaparalogs belong to group II. We have identified a fourthGiardiacytb5paralog (gCYTb5-IV) that is rather divergent and possesses an unusual 134-residue N-terminal extension. RecombinantGiardiacytb5proteins, including gCYTb5-IV, were expressed inEscherichia coliand exhibited characteristic UV-visible spectra that corresponded to heme-loaded cytb5proteins. The expression of the recombinant gCYTb5-IV inG. intestinalisresulted in the increased import of extracellular heme and its incorporation into the protein, whereas this effect was not observed when gCYTb5-IV containing a mutated heme-binding site was expressed. The electrons forGiardiacytb5proteins may be provided by the NADPH-dependent Tah18-like oxidoreductase GiOR-1. Therefore, GiOR-1 and cytb5may constitute a novel redox system inG. intestinalis. To our knowledge,G. intestinalisis the first anaerobic eukaryote in which the presence of heme has been directly demonstrated.