Structural features promoting dioxygen production by Dechloromonas aromatica chlorite dismutase

Abstract

Chlorite dismutase (Cld) is a heme enzyme capable of rapidly and selectively decomposing chlorite (ClO2−) to Cl− and O2. The ability of Cld to promote O2 formation from ClO2− is unusual. Heme enzymes generally utilize ClO2− as an oxidant for reactions such as oxygen atom transfer to, or halogenation of, a second substrate. The X-ray crystal structure of Dechloromonas aromatica Cld co-crystallized with the substrate analogue nitrite (NO2−) was determined to investigate features responsible for this novel reactivity. The enzyme active site contains a single b-type heme coordinated by a proximal histidine residue. Structural analysis identified a glutamate residue hydrogen-bonded to the heme proximal histidine that may stabilize reactive heme species. A solvent-exposed arginine residue likely gates substrate entry to a tightly confined distal pocket. On the basis of the proposed mechanism of Cld, initial reaction of ClO2− within the distal pocket generates hypochlorite (ClO−) and a compound I intermediate. The sterically restrictive distal pocket probably facilitates the rapid rebound of ClO− with compound I forming the Cl− and O2 products. Common to other heme enzymes, Cld is inactivated after a finite number of turnovers, potentially via the observed formation of an off-pathway tryptophanyl radical species through electron migration to compound I. Three tryptophan residues of Cld have been identified as candidates for this off-pathway radical. Finally, a juxtaposition of hydrophobic residues between the distal pocket and the enzyme surface suggests O2 may have a preferential direction for exiting the active site.

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.…”

“…However, in most haeme proteins the Fe-proximal His represents the only covalent bond between the haeme chromophore and the protein, and stable incorporation of the prosthetic group occurs via several non-covalent interactions. These include H-bonding networks at the proximal side [54] as shown for haeme b-containing Clds [12,20,30,55].…”

“…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.…”

The Chlorite Dismutase (HemQ) from Staphylococcus aureus Has a Redox-sensitive Heme and Is Associated with the Small Colony Variant Phenotype

“…By contrast, holo-HemQ has an apparent molecular mass of ϳ187 kDa, consistent with a homohexamer. Analytical gel filtration carried out in a similar fashion for DaCld suggested a homotetramer in solution; however, crystallography showed the functional protein to be a pentamer (4,7). Other Cld-family proteins have exhibited a similar level of discrepancy between gel filtration estimates and crystallographic measurements of oligomerization states (11,49,50).…”

Unexpected Diversity of Chlorite Dismutases: a Catalytically Efficient Dimeric Enzyme from Nitrobacter winogradskyi

“…Cld is a heme enzyme that, based on the published structural information, consists of five or six identical subunits (11,12,16,22). Besides its importance for bioengineering, Cld is extremely interesting from a biochemical perspective.…”