Compound I Formation and Reactivity in Dimeric Chlorite Dismutase: Impact of pH and the Dynamics of the Catalytic Arginine

Abstract

The heme enzyme chlorite dismutase (Cld) catalyzes the degradation of chlorite to chloride and dioxygen. Many questions about the molecular reaction mechanism of this iron protein have remained unanswered, including the electronic nature of the catalytically relevant oxoiron(IV) intermediate and its interaction with the distal, flexible, and catalytically active arginine. Here, we have investigated the dimeric Cld from Cyanothece sp. PCC7425 (CCld) and two variants having the catalytic arginine R127 (i) hydrogen-bonded to glutamine Q74 (wild-type CCld), (ii) arrested in a salt bridge with a glutamate (Q74E), or (iii) being fully flexible (Q74V). Presented stopped-flow spectroscopic studies demonstrate the initial and transient appearance of Compound I in the reaction between CCld and chlorite at pH 5.0 and 7.0 and the dominance of spectral features of an oxoiron(IV) species (418, 528, and 551 nm) during most of the chlorite degradation period at neutral and alkaline pH. Arresting the R127 in a salt bridge delays chlorite decomposition, whereas increased flexibility accelerates the reaction. The dynamics of R127 does not affect the formation of Compound I mediated by hypochlorite but has an influence on Compound I stability, which decreases rapidly with increasing pH. The decrease in activity is accompanied by the formation of protein-based amino acid radicals. Compound I is demonstrated to oxidize iodide, chlorite, and serotonin but not hypochlorite. Serotonin is able to dampen oxidative damage and inactivation of CCld at neutral and alkaline pH. Presented data are discussed with respect to the molecular mechanism of Cld and the pronounced pH dependence of chlorite degradation.

Tyr 118-Mediated Electron Transfer is Key to the Chlorite Decomposition in Heme-Dependent Chlorite Dismutase

“…Interestingly, the Cl–O Fe bond cleavage undergoes via a homolytic cleavage, generating a ClO Cl • radical and a Fe­(III)–O Fe H heme species. This finding is in accord with a much recent study, where a ClO Cl • radical formation is proposed during decomposition by Cld. The barrier for this step is smaller for 6 TS1, i.e., 7.8 kcal/mol (see Figure a for barriers).…”

“…However, our study also supports the homolytic cleavage, in contrast to the study by Püschmann et al In addition, our study highlights a key role of Y118 in ET that reduces the Fe III → Fe II and triggers a homolytic cleavage of the chlorite ion. Our mechanistic study shows that there is a spin crossover between the double and sextet state which is also well supported by recent EPR studies. , As such, this study would help in understanding the mechanism of chlorite decomposition more clearly and provides crucial information about amino acids participating in the ET process. We believe that such a mechanistic study of the chlorite decomposition could provide a crucial lead in further bioengineering the Cld enzyme for enhancing the efficiency.…”

“…• radical and a Fe(III)− O Fe H heme species. This finding is in accord with a much recent study, 37 where a ClO Cl…”

“…Interestingly, this helix is associated with a loop that contains W227 which comes closer to the heme center due to this conformational rearrangement (see Figures and S3 in Supporting Information for the variation of distance with simulation time) and thereby forms an interaction with the axial ligand, i.e., H170 of heme porphyrin. It is supposed that the hexacoordinated heme porphyrin after the binding of chlorite and axial histidine ligand must be stabilized by a polar residue; therefore, we believe W227 must be playing a similar role. Also, according to a study, a mutation of W227 → F does not show loss of activity, and it retains many of the properties of wild-type enzyme such as the formation of protein-based cation radical with PAA (peracetic acid).…”

“…Our mechanistic study shows that there is a spin crossover between the double and sextet state which is also well supported by recent EPR studies. 9,37 As such, this study would help in understanding the mechanism of chlorite decomposition more clearly and provides crucial information about amino acids participating in the ET process. We believe that such a mechanistic study of the chlorite decomposition could provide a crucial lead in further bioengineering the Cld enzyme for enhancing the efficiency.…”