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Review
. 2007 Jul;40(7):475-83.
doi: 10.1021/ar700052v. Epub 2007 Jun 14.

Finding intermediates in the O2 activation pathways of non-heme iron oxygenases

E G Kovaleva  1 , M B Neibergall, S Chakrabarty, J D Lipscomb
Affiliations
Review

Finding intermediates in the O2 activation pathways of non-heme iron oxygenases

E G Kovaleva et al. Acc Chem Res. 2007 Jul.

Abstract

Intermediates in the reaction cycle of an oxygenase are usually very informative with respect to the chemical mechanism of O 2 activation and insertion. However, detection of these intermediates is often complicated by their short lifetime and the regulatory mechanism of the enzyme designed to ensure specificity. Here, the methods used to detect the intermediates in an extradiol dioxygenase, a Rieske cis-dihydrodiol dioxygenase, and soluble methane monooxygenase are discussed. The methods include the use of alternative, chromophoric substrates, mutagenesis of active site catalytic residues, forced changes in substrate binding order, control of reaction rates using regulatory proteins, and initialization of catalysis in crystallo.

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Figures

FIGURE 1
FIGURE 1
Proposed extradiol dioxygenase mechanism. R = -CH2COOH (HPCA) or –NO2 (4NC) for the studies described here. Single hydroxyl deprotonation during binding results in a bound monoanion in the case of HPCA (shown) and a bound dianion for 4NC.
FIGURE 2
FIGURE 2
Intermediates discovered in the reaction cycle of the H200N mutant of HPCD when using 4NC as the substrate (rate constants at 4 °C, pH 7.5). The substrate binding steps occur at similar rates for the wild type enzyme. The H200N reaction diverges from the normal cycle after the alkylperoxo intermediate to yield a quinone rather than a ring open product. When using HPCA, the normal ring-open product is obtained. (See references 10 and11 for experimental conditions)
FIGURE 3
FIGURE 3
Oxy-intermediate formation by the H200N mutant of HPCD using HPCA as the substrate. Inset: O2 concentration dependence of the intermediate formation.
FIGURE 4
FIGURE 4
X-ray crystal structure of the intermediates of the HPCD reaction cycle. PDB files: E (1F1X), ES (1Q0C), other intermediates (2IGA). Inset: The aromatic ring of 4NC becomes progressively less planar as the E-semiquinone-superoxo and the alkylperoxo intermediates form. The rightmost structure is the oxidized H200E mutant complex (unpublished data).
FIGURE 5
FIGURE 5. Single turnover (STO) and peroxide shunt reaction cycles of NDO.
FIGURE 6
FIGURE 6
The peroxide shunt reaction of BZDO is slowed by combination of substrates with incorrect forms of the enzyme.
FIGURE 7
FIGURE 7
EPR (top) spectra of intermediates in the peroxide shunt of BZDO reacting with benzoate. Mössbauer spectrum and simulation (red) of the nearly EPR silent intermediate after 4 min of reaction. Adapted from reference .
FIGURE 8
FIGURE 8
X-ray crystal structure of the oxy-intermediate of NDO (PDB 1O7N).
FIGURE 9
FIGURE 9
Intermediates in the reaction cycle of sMMO.
FIGURE 10
FIGURE 10
Proposed structure of sMMO P and Q from spectroscopic studies. Although P is clearly a peroxy adduct, its precise structure has not been definitively established.
FIGURE 11
FIGURE 11
Molecular sieve model for the methane selectivity of MMOH. Adapted from reference .
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References

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