Understanding the roles of strictly conserved tryptophan residues in O₂producing chlorite dismutases

Abstract

The chlorite dismutases (Clds) degrade ClO2− to O2 and Cl− in perchlorate respiring bacteria, and they serve still poorly defined cellular roles in other diverse microbes. These proteins share 3 highly conserved Trp residues, W155, W156, and W227, on the proximal side of the heme. The Cld from Dechloromonas aromatica (DaCld) has been shown to form protein-based radicals in its reactions with ClO2− and peracetic acid. The roles of the conserved Trp residues in radical generation and in enzymatic function were assessed via spectroscopic and kinetic analysis of their Phe mutants. The W155F mutant was the most dramatically affected, appearing to lose the characteristic pentameric oligomerization state, secondary structure, and heme binding properties of the WT protein. The W156F mutant initially retains many features of the WT protein but over time acquires many of the features of W155F. Conversion to an inactive, heme-free form is accelerated by dilution, suggesting loss of the protein’s pentameric state. Hence, both W155 and W156 are important for heme binding and maintenance of the protein’s reactive pentameric structure. W227F by contrast retains many properties of the WT protein. Important differences are noted in the transient kinetic reactions with peracetic acid (PAA), where W227F appears to form an [Fe(IV)=O]-containing intermediate, which subsequently converts to an uncoupled [Fe(IV)=O + AA+.] system in a [PAA]-dependent manner. This is in contrast to the peroxidase-like formation of [Fe(IV)=O] coupled to a porphyrin π-cation radical in the WT protein, which decays in a [PAA]-independent manner. These observations and the lack of redox protection for the heme in any of the Trp mutants suggests a tendency for protein radical formation in DaCld that is independent of any of these conserved active site residues.

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

“…For another functional chlorite dismutase, namely DaCld, the observed ν(Fe-Im) stretching band (222 cm − 1 ) suggests weaker binding between the haeme iron and its proximal ligand compared with NdCld [47]. Even though no direct data concerning the stability of DaCld are available, indirect methods and observations support a lower conformational and thermal stability of DaCld compared with NdCld [24,27,30]. Retrospectively, this correlates with the observed ν(Fe-Im) stretching frequency of both functional Clds.…”

“…Studies of proximal residue mutants of DaCld and NdCld revealed that manipulation of the rigid H-bonding network in Cld significantly weakens binding of haeme b and thereby destabilizes the protein [25][26][27][28][29][30]. In the present work, we studied the effect of disrupting the proximal H-bonding network by comparing wild-type NdCld with the proximal variants E210A and K141E.…”

Peroxidase-Type Reactions Suggest a Heterolytic/Nucleophilic O–O Joining Mechanism in the Heme-Dependent Chlorite Dismutase

“…24, 35, 36 The Da Cld crystal structure shows three highly conserved Trp and two Tyr residues within 5 Å of the heme (Scheme 2), any or all of which might in principle provide a conduit for radical migration. 62 The observed reaction of [Fe IV =O] + (AA•) with a single equivalent of ascorbate to yield a ferric species would then most likely be due to selective one-electron reduction of the ferryl iron to the ferric. This might occur if the protein radical species, which one would expect to have proceeded out of the active site, were inaccessible to the reductant.…”

Production of Dioxygen in the Dark: Dismutases of Oxyanions

“…Finally, a second tryptophan near the heme’s periphery (Trp156) and two residues from a triad of hydrophobic amino acids above the heme plane (Leu185 and Phe200) are strictly conserved among all Clds. The tryptophan appears to be critical for maintaining the bound heme as well as the protein’s oligomeric state [79], and hydrophobicity in the distal pocket may be essential for stabilizing reaction intermediates (see Section 4.2. )…”

“…Indeed, a Compound I species prepared in the stopped flow did not react with exogenously supplied ClO − /HClO to form O 2 , as the reaction appears to be kinetically out-competed by decay of the Compound I intermediate [89]. Though the pathway has not been studied, Compound I’s breakdown has been presumed to occur via the conserved tyrosines that ring (and in one case form hydrogen bonds with) the heme [79]. …”