An interaction domain drives heme exchange in the C. albicans CFEM hemophores cascade

The fungal pathobiont Candida albicans acquires heme from host proteins via a set of soluble and anchored extracellular CFEM (Conserved in Fungal Extracellular Membrane)-type hemophores that can capture the bound heme and exchange it, eventually delivering it to the cell membrane for endocytosis into the yeast cell. Yet the molecular mechanism by which the heme is transferred through this protein cascade across the cell envelope remains unclear. To address this mechanism, we developed a set of fusions of three C. albicans hemophores with fluorescent proteins. Fluorescence of these fusion proteins is strongly quenched when heme is bound to the hemophore moiety, enabling instantaneous measurement of heme transfer. Kinetic analysis of the different transfer reactions reveals that heme transfer from the host protein to the CFEM hemophores and between the CFEM hemophores are governed by different regimes. Kinetics of heme transfer from hemoglobin or serum albumin to the CFEM hemophores is mainly first-order, suggesting that heme stochastically released from host proteins is captured by the hemophores. In contrast, exchange of heme between the hemophores is near second-order, consistent with a mechanism requiring protein-protein interactions. We do in fact find that CFEM hemophores can interact in homodimeric and heterodimeric complexes, and identify and mutagenize the interaction surface. While the dimerization-defective mutants of the soluble hemophore Csa2 are proficient in heme binding and extraction, they are defective in heme exchange, supporting a model of heme transfer by direct interaction between the members of the fungal hemophore cascade.