10.1021/acs.biochem.5b00666.s001 Elizabeth B. Draganova Elizabeth B. Draganova Neval Akbas Neval Akbas Seth A. Adrian Seth A. Adrian Gudrun S. Lukat-Rodgers Gudrun S. Lukat-Rodgers Daniel P. Collins Daniel P. Collins John H. Dawson John H. Dawson Courtni E. Allen Courtni E. Allen Michael P. Schmitt Michael P. Schmitt Kenton R. Rodgers Kenton R. Rodgers Dabney W. Dixon Dabney W. Dixon Heme Binding by <i>Corynebacterium diphtheriae</i> HmuT: Function and Heme Environment American Chemical Society 2015 WT heme pocket residue mutants Y 235 side Heme EnvironmentThe heme uptake pathway H 136A Y 235A hydrogen bond acceptor Hemoglobin utilization assays ferric HmuT H 136 M 292A Characterization Y 235 ligand Corynebacterium diphtheriae HmuT hydrogen bond donor H 136A HmuT FeCO Raman shifts Ferrous Y 235A ABC transporter HmuUV Y 235A H 136 side 2015-11-03 00:00:00 Journal contribution https://acs.figshare.com/articles/journal_contribution/Heme_Binding_by_i_Corynebacterium_diphtheriae_i_HmuT_Function_and_Heme_Environment/2115688 The heme uptake pathway (hmu) of <i>Corynebacterium diphtheriae</i> utilizes multiple proteins to bind and transport heme into the cell. One of these proteins, HmuT, delivers heme to the ABC transporter HmuUV. In this study, the axial ligation of the heme in ferric HmuT is probed by examination of wild-type (WT) HmuT and a series of conserved heme pocket residue mutants, H136A, Y235A, and M292A. Characterization by UV–visible, resonance Raman, and magnetic circular dichroism spectroscopies indicates that H136 and Y235 are the axial ligands in ferric HmuT. Consistent with this assignment of axial ligands, ferric WT and H136A HmuT are difficult to reduce while Y235A is reduced readily in the presence of dithionite. The FeCO Raman shifts in WT, H136A, and Y235A HmuT–CO complexes provide further evidence of the axial ligand assignments. Additionally, these frequencies provide insight into the nonbonding environment of the heme pocket. Ferrous Y235A and the Y235A–CO complex reveal that the imidazole of H136 exists in two forms, one neutral and one with imidazolate character, consistent with a hydrogen bond acceptor on the H136 side of the heme. The ferric fluoride complex of Y235A reveals the presence of at least one hydrogen bond donor on the Y235 side of the heme. Hemoglobin utilization assays showed that the axial Y235 ligand is required for heme uptake in HmuT.