Spectroscopic characterization and ligand‐binding properties of chlorite dismutase from the chlorate respiring bacterial strain GR‐1

Abstract

Chlorite dismutase (EC 1.13.11.49), an enzyme capable of reducing chlorite to chloride while producing molecular oxygen, has been characterized using EPR and optical spectroscopy. The EPR spectrum of GR-1 chlorite dismutase shows two different high-spin ferric heme species, which we have designated ÔnarrowÕ (g x,y,z ¼ 6.24, 5.42, 2.00) and ÔbroadÕ (g z,y,x ¼ 6.70, 5.02, 2.00). Spectroscopic evidence is presented for a proximal histidine co-ordinating the heme iron center of the enzyme. The UV/visible spectrum of the ferrous enzyme and EPR spectra of the ferric hydroxide and imidazole adducts are characteristic of a heme protein with an axial histidine co-ordinating the iron. Furthermore, the substrate analogs nitrite and hydrogen peroxide have been found to bind to ferric chlorite dismutase. EPR spectroscopy of the hydrogen peroxide adduct shows the loss of both high-spin and low-spin ferric signals and the appearance of a sharp radical signal. The NO adduct of the ferrous enzyme exhibits a low-spin EPR signal typical of a five-co-ordinate heme iron nitrosyl adduct. It seems that the bond between the proximal histidine and the iron is weak and can be broken upon binding of NO. The midpoint potential, E m (Fe 3+/2+ ) ¼ )23 mV, of chlorite dismutase is higher than for most heme enzymes. The spectroscopic features and redox properties of chlorite dismutase are more similar to the gas-sensing hemoproteins, such as guanylate cyclase and the globins, than to the heme enzymes.

The homopentameric chlorite dismutase from Magnetospirillum sp.

“…The iron atom of the heme cofactor is pentacoordinated with the axial position occupied by the imidazole ring of one His residue, in agreement with the high-spin Fe(III) EPR signal observed in the as-isolated Cld samples at pH 6.0-7.0 [15,22].…”

“…aromatica RCB in their as-prepared forms at pH 5.0-7.0 are straightforward to explain [8,15,22]. In these Cld, the EPR signal observable at liquid helium temperatures arises from one high-spin Fe 3+ -heme species corresponding to the pentacoordinated ferric ion present in the Cld active site.…”

Crystal Structure of Chlorite Dismutase, a Detoxifying Enzyme Producing Molecular Oxygen

“…30 Redox titration resulted in the following reduction potentials of the different heme species: − 158 ± 9 mV for the five-coordinate Cld, − 170 ± 6 mV for imidazole-bound Cld, and + 22 ± 6 mV for the putative protonated imidazole-bound Cld. The reduction potential for the recombinant Cld is 135 mV lower than that for the wild-type enzyme, 8 which could be caused by subtle structural differences in the proximity of the heme. The reduction potential of heme iron in enzymes with a proximal histidine ligand is modulated by the basicity of the proximal histidine.…”

“…6 Cld is a multimeric protein containing one iron protoheme IX per 28-kDa subunit. 7,8 Intriguing properties of this enzyme are the extraordinary specificity for chlorite and the efficiency of the chlorite conversion. Moreover, Cld is currently the only known heme-based enzyme that is able to perform O-O bond-formation catalysis as its primary function.…”

From chlorite dismutase towards HemQ–the role of the proximal H-bonding network in haeme binding

“…This includes Clds from perchlorate reducing bacteria (Azospira oryzae GR-1, Ideonella dechloratans, Dechloromonas aromatica, Pseudomonas chloritidismutans, Magnetospirillum sp.) [13][14][15][16][17][18], nitrite-oxidizing bacteria ('Candidatus Nitrospira defluvii', Nitrobacter winogradskyi) [12,19,20], from cyanobacteria (e.g. Cyanothece sp.…”