The Oxygen Dilemma: A Severe Challenge for the Application of Monooxygenases?

Abstract

Monooxygenases are promising catalysts because they in principle enable the organic chemist to perform highly selective oxyfunctionalisation reactions that are otherwise difficult to achieve. For this, monooxygenases require reducing equivalents, to allow reductive activation of molecular oxygen at the enzymes' active sites. However, these reducing equivalents are often delivered to O2 either directly or via a reduced intermediate (uncoupling), yielding hazardous reactive oxygen species and wasting valuable reducing equivalents. The oxygen dilemma arises from monooxygenases' dependency on O2 and the undesired uncoupling reaction. With this contribution we hope to generate a general awareness of the oxygen dilemma and to discuss its nature and some promising solutions.

Keywords: biocatalysis; monooxygenases; oxidoreductases; oxyfunctionalization; uncoupling.

Scheme 1

The synthetic scope of monooxygenases. Within the enzymes' active sites, highly reactive oxygen‐transfer species are generated through reductive activation of molecular oxygen. The protein scaffold controls the interaction between these reactive oxygen transfer species [e.g., Fe⋅oxo complexes or (hydro)peroxyflavins] and the substrates, leading to highly selective oxyfunctionalisation reactions.1c

Scheme 2

Simplified mechanism of flavin‐dependent monooxygenases, consisting of a) NAD(P)H‐dependent reduction of the flavin prosthetic group, followed by b) activation of molecular oxygen as a (hydro)peroxyflavin, and c) substrate oxygenation. The catalytic cycle is closed after d) elimination of water and reformation of the oxidised flavin. Alternatively, e) the (hydro)peroxyflavin can eliminate H₂O₂ spontaneously (uncoupling reaction).

Scheme 3

Simplified mechanism of haem‐dependent monooxygenases, consisting of two single‐electron transfer steps to haem iron (steps b and d) and formation of “compound I” (step f) to perform the oxygenation reaction (step g). The elimination of H₂O₂ from the intermediate Fe^III peroxo species is shown in step h.

Scheme 4

Simplified molecular architecture of multicomponent monooxygenases that are not directly dependent on NAD(P)H. Blue: path of reducing equivalents. NAD(P)H serves as a general reductant, transferring its reducing equivalents to a mediator molecule (either a flavin or an iron–sulfur cluster protein) with catalysis by a reductase. The usually protein‐based mediator delivers the reducing equivalents to the monooxygenase subunit for productive oxygen activation. However, direct reaction of the reduced mediator with O₂ leads to futile reoxidation and (eventually) H₂O₂ formation.

Scheme 5

Molecular oxygen (two spins) can react quickly with single‐electron mediators because the sum of spins does not change during the reaction (spin‐allowed reaction). Hydride donors (or two‐electron mediators in general) react with molecular oxygen far more slowly because the sum of spins changes during the reaction (spin‐forbidden reaction).

Figure 1

Comparison of the reduction kinetics of NAD⁺ (black) and CytC (red) with [Cp*Rh(bpy)H]⁺ in the presence of O₂. Comparative experiments with a Clark electrode demonstrated that the dissolved O₂ remained constant in the case of NAD⁺ reduction whereas it decreased to less than 10 μm in the first 30 s of the CytC reduction experiment.

Scheme 6

Classification of P450 monooxygenases. Fd: Ferredoxin. CPR: cytochrome P450 reductase.

Scheme 7

Simplified regeneration of (mono)oxygenases by direct reductive regeneration of the enzymes' active sites.

Scheme 8

Covalent linkage of a Ru‐photosensitzer/mediator to a P450 monooxygenase to facilitate electron transfer.

Scheme 9

5‐Deazaflavins (bottom) as O₂‐stable reduced mediators (in comparison with normal flavins, top).

Scheme 10

Simplified scheme for peroxygenase‐catalysed oxyfunctionalisation reactions exploiting the oxygen dilemma.