Charge Maintenance during Catalysis in Nonheme Iron Oxygenases

Abstract

Here, the choice of the first coordination shell of the metal center is analyzed from the perspective of charge maintenance in a binary enzyme-substrate complex and an O₂-bound ternary complex in the nonheme iron oxygenases. Comparing homogentisate 1,2-dioxygenase and gentisate dioxygenase highlights the significance of charge maintenance after substrate binding as an important factor that drives the reaction coordinate. We then extend the charge analysis to several common types of nonheme iron oxygenases containing either a 2-His-1-carboxylate facial triad or a 3-His or 4-His ligand motif, including extradiol and intradiol ring-cleavage dioxygenases, thiol dioxygenases, α-ketoglutarate-dependent oxygenases, and carotenoid cleavage oxygenases. After forming the productive enzyme-substrate complex, the overall charge of the iron complex at the 0, +1, or +2 state is maintained in the remaining catalytic steps. Hence, maintaining a constant charge is crucial to promote the reaction of the iron center beginning from the formation of the Michaelis or ternary complex. The charge compensation to the iron ion is tuned not only by protein-derived carboxylate ligands but also by substrates. Overall, these analyses indicate that charge maintenance at the iron center is significant when all the necessary components form a productive complex. This charge maintenance concept may apply to most oxygen-activating metalloenzymes systems that do not draw electrons and protons step-by-step from a separate reactant, such as NADH, via a reductase. The charge maintenance perception may also be useful in proposing catalytic pathways or designing prototypical reactions using artificial or engineered enzymes for biotechnological applications.

Keywords: biocatalysis; charge compensation; electron transfer; iron catalysts; oxidation; oxygenation.

Figure 1.

Comparison of the chemical reactions and the catalytic iron centers of HGDO and salicylate 1,2-dioxygenase (SDO), a GDO homologue. (A) The top panel shows HGDO catalyzing the oxidation of homogentisate to maleylacetoacetate, and the bottom panel depicts the active site from HGDO found in Pseudomonas putida (PDB ID 4AQ6), which has a bound homogentisate. (B) The top panel shows GDO catalyzing the oxidation of gentisate, and the bottom panel depicts the active site of SDO (PDB ID 3NL1), which has a bound gentisate. The residues ligated to the Fe(II) ion (brown) are drawn in gray. The substrate of the respective enzyme is colored yellow and represented as sticks and balls.

Figure 2.

Structural snapshots of the HAO-mediated dioxygenation with its native substrate. Crystal structures of (I) the enzyme alone (PDB ID 4R52), (II) the monodentate binding of the substrate (6VI6), (III) the bidentate binding of the substrate in the ES complex (6VI7), (IV) the superoxo intermediate (6VI8), (V) alkylperoxo (6VI9), (VI) the monooxygenated lactone intermediate (6VIA), (VII) ACMS in the 3E,5Z,2t,4c-enol tautomer conformation (6X11), and (VIII) the dioxygenation product ACMS in the 3E,5Z,2t,4t-enol tautomer conformation (6VIB). Solvent-derived ligands are shown in cyan. Substrate carbons are shown in bright yellow, and carbons in protein ligands are shown in gray. Oxygen and nitrogen are shown in red and blue, respectively.

Figure 3.

Active sites of (A) TauD (PDB ID 1GY9) and (B) SyrB2 (PDB ID 2FCT). Protein ligand residues coordinated to the Fe(II) ion center (brown) are drawn in gray with the cosubstrate, and αKG is drawn in yellow as sticks and balls. The chloride ion, which provides a negative charge to the iron, is drawn as a green sphere.

Scheme 1.

Mechanism of Human Homogentisate 1,2-Dioxygenase (HGDO)^{a a}A key intermediate, a productive iron complex with a neutral charge, is highlighted with a light green background for comparison with the various catalytic systems discussed in this perspective. A negatively charged donor ligand is featured in purple. The overall formal charge of the iron center is indicated outside the brackets.

Scheme 2.

Hypothesized Intermediates of Gentisate 1,2-Dioxygenase (GDO) and Salicylate 1,2-Dioxygenase (SDO)^{a a}R = OH (gentisate) or H (salicylate).

Scheme 3.

(A) Intradiol and (B) Extradiol Dioxygenase Degradation of Catechols^{a a}The red C=C double bond denotes the bond cleavage site.

Scheme 4.

Mechanism of Intradiol Dioxygenases as Illustrated by Protocatechuate 3,4-Dioxygenase (PCD)

Scheme 5.

General Mechanism for Extradiol Dioxygenases as Illustrated by HPCD

Scheme 6.

Proposed Models of CDO Catalyzing the Oxidation of Cysteine to Cysteinesulfinate^{a a}Pathway A is the simultaneous delivery of O₂ to the substrate thiol group, whereas pathway B is a stepwise model where the distal O atom is the first to transfer.

Scheme 7.

General Sulfoxide Synthase Reactions of (A) OvoA and (B) EgtB

Scheme 8.

Generic Mechanism of an αKG-Dependent Dioxygenase Performing Hydroxylation on Its Substrate

Scheme 9.

Proposed Model of CCOs Performing Oxygenation on a Substrate