Chlorite dismutase (Cld) catalyzes the reduction of chlorite to chloride and dioxygen. Here, the ligand binding to Cld of Magnetospirillum sp. (MaCld) is investigated with X-ray crystallography and electron paramagnetic resonance (EPR). EPR reveals a large heterogeneity in the structure of wild-type MaCld, showing a variety of low- and high-spin ferric heme forms. Addition of an axial ligand, such as azide or imidazole, removes this heterogeneity almost entirely. This is in line with the two high resolution crystal structures of MaCld obtained in the presence of azide and thiocyanate that show the coordination of the ligands to the heme iron. The crystal structure of the MaCld-azide complex reveals a single well-defined orientation of the azide molecule in the heme pocket. EPR shows, however, a pH-dependent heme structure, probably due to acid-base transitions of the surrounding amino-acid residues stabilizing azide. For the azide and imidazole complex of MaCld, the hyperfine and nuclear quadrupole interactions with the close-by (14)N and (1)H nuclei are determined using pulsed EPR. These values are compared to the corresponding data for the low-spin forms observed in the ferric wild-type MaCld and to existing EPR data on azide and imidazole complexes of other heme proteins.

The cytoglobins of the Antarctic fish Chaenocephalus aceratus and Dissostichus mawsoni have many features in common with human cytoglobin. These cytoglobins are heme proteins in which the ferric and ferrous forms have a characteristic hexacoordination of the heme iron, i.e. axial ligation of two endogenous histidine residues, as confirmed by electron paramagnetic resonance, resonance Raman and optical absorption spectroscopy. The combined spectroscopic analysis revealed only small variations in the heme-pocket structure, in line with the small variations observed for the redox potential. Nevertheless, some striking differences were also discovered. Resonance Raman spectroscopy showed that the stabilization of an exogenous heme ligand, such as CO, occurs differently in human cytoglobin in comparison with Antarctic fish cytoglobins. Furthermore, while it has been extensively reported that human cytoglobin is essentially monomeric and can form an intramolecular disulfide bridge that can influence the ligand binding kinetics, 3D modeling of the Antarctic fish cytoglobins indicates that the cysteine residues are too far apart to form such an intramolecular bridge. Moreover, gel filtration and mass spectrometry reveal the occurrence of non-covalent multimers (up to pentamers) in the Antarctic fish cytoglobins that are formed at low concentrations. Stabilization of these oligomers by disulfide-bridge formation is possible, but not essential. If intermolecular disulfide bridges are formed, they influence the heme-pocket structure, as is shown by EPR measurements.

Antioxidants and Redox SignalingStructural and functional characterization of the globin-coupled sensors of Azotobacter vinelandii and Bordetella pertussis

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