HupZ is an expected heme degrading enzyme in the heme acquisition and utilization pathway in Group A Streptococcus. The isolated HupZ protein containing a C-terminal V5-His6 tag exhibits a weak heme degradation activity. Here, we revisited and characterized the HupZ-V5-His6 protein via biochemical, mutagenesis, protein quaternary structure, UV–vis, EPR, and resonance Raman spectroscopies. The results show that the ferric heme-protein complex did not display an expected ferric EPR signal and that heme binding to HupZ triggered the formation of higher oligomeric states. We found that heme binding to HupZ was an O2-dependent process. The single histidine residue in the HupZ sequence, His111, did not bind to the ferric heme, nor was it involved with the weak heme-degradation activity. Our results do not favor the heme oxygenase assignment because of the slow binding of heme and the newly discovered association of the weak heme degradation activity with the His6-tag. Altogether, the data suggest that the protein binds heme by its His6-tag, resulting in a heme-induced higher-order oligomeric structure and heme stacking. This work emphasizes the importance of considering exogenous tags when interpreting experimental observations during the study of heme utilization proteins.

The UV–vis absorption, Raman imaging, and resonance Raman (rR) spectroscopy methods were employed to study cyanohemoglobin (HbCN) adducts inside living functional red blood cells (RBCs). The cyanide ligands are especially optically sensitive probes of the active site environment of heme proteins. The rR studies of HbCN and its isotopic analogues ( 13 CN – , C 15 N – , and 13 C 15 N – ), as well as a careful deconvolution of spectral data, revealed that the ν(Fe–CN) stretching, δ(Fe–CN) bending, and ν(C≡N) stretching modes occur at 454, 382, and 2123 cm –1 , respectively. Interestingly, while the ν(Fe–CN) modes exhibit the same frequencies in both the isolated and RBC-enclosed hemoglobin molecules, small frequency differences are observed in the δ(Fe–CN) bending modes and the values of their isotopic shifts. These studies show that even though the overall tilted conformation of the Fe–C≡N fragment in the isolated HbCN is preserved in the HbCN enclosed within living cells, there is a small difference in the degree of distortion of the Fe–C≡N fragment. The slight changes in the ligand geometry can be reasonably attributed to the high ordering and tight packing of Hb molecules inside RBCs.

Human heme oxygenase (hHO-1) is a physiologically important enzyme responsible for free heme catabolism. The enzyme’s high regiospecificity is controlled by the distal site hydrogen bond network that involves water molecules and the D140 amino acid residue. In this work, we probe the active site environment of the wild-type (WT) hHO-1 and its D140 mutants using resonance Raman (rR) spectroscopy. Cyanide ligands are more stable than dioxygen adducts and are an effective probe of active site environment of heme proteins. The inherently linear geometry of the Fe–C–N fragment can be altered by the steric, electrostatic, and H-bonding interactions imposed by the amino acid residues present in the heme distal site, resulting in a tilted or bent configuration. The WT hHO-1 and its D140A, D140N, and D140E mutants were studied in the presence of natural abundance CN– and its isotopic analogues (13CN–, C15N–, and 13C15N–). Deconvolution of spectral data revealed that the ν­(Fe–CN) stretching and δ­(Fe–CN) bending modes are present at 454 and 376 cm–1, respectively. The rR spectral patterns of the CN– adducts of WT revealed that the Fe–C–N fragment adopts a tilted conformation, with a larger bending contribution for the D140A, D140N, and D140E mutants. These studies suggest that the FeCN fragment in hHO-1 is tilted more strongly toward the porphyrin macrocycle compared to other histidine-ligated proteins, reflecting the propensity of the exogenous hHO-l ligands to position toward the α-meso-carbon, which is crucial for the HO reactivity and essential for regioselectivity.

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