Enzymatic Hydroxylation of Aliphatic C–H Bonds by a Mn/Fe Cofactor

Abstract

The aerobic oxidation of carbon−hydrogen (C−H) bonds in biology is currently known to be accomplished by a limited set of cofactors that typically include heme, nonheme iron, and copper. While manganese cofactors perform difficult oxidation reactions, including water oxidation within Photosystem II, they are generally not known to be used for C−H bond activation, and those that do catalyze this important reaction display limited intrinsic reactivity. Here we report that the 2-aminoisobutyric acid hydroxylase from Rhodococcus wratislaviensis, AibH1H2, requires manganese to functionalize a strong, aliphatic C−H bond (BDE = 100 kcal/mol). Structural and spectroscopic studies of this enzyme reveal a redox-active, heterobimetallic manganese−iron active site at the locus of O 2 activation and substrate coordination. This result expands the known reactivity of biological manganese−iron cofactors, which was previously restricted to single-electron transfer or stoichiometric protein oxidation. Furthermore, the AibH1H2 cofactor is supported by a protein fold distinct from typical bimetallic oxygenases, and bioinformatic analyses identify related proteins abundant in microorganisms. This suggests that many uncharacterized monooxygenases may similarly require manganese to perform oxidative biochemical tasks.

Bioinformatic Discovery of a Cambialistic Monooxygenase

“…This hypothesis is bolstered by the spectrum of 57 Fe-labeled Mn/Fe-SfbO (Figure B) which can be simulated with the inclusion of an additional 57 Fe hyperfine coupling tensor ( A Fe [(−70 −70 −70) MHz]). These EPR signals are comparable to those of related cofactors found in AibH1H2, class Ic ribonucleotide reductases, and R2Lox, which harbor antiferromagnetically coupled Mn/Fe clusters. The addition of either 300 mM sodium sulfate (Figure C), or 200 mM PIPES (Figure D) to similarly prepared Mn/Fe-SfbO samples resulted in distinct EPR signals.…”

“…This ion appears to be tightly bound via strong electrostatic and hydrogen-bonding interactions with 67 N, 232 R, 235 R, 280 Y, and 341 W, and this observation may provide insight into functional groups present on the natural substrate ( vide infra ). Excepting the sulfate-binding interactions found in SfbO, the active site structure closely mimics that found in AibH2, PtmU3 (Figure S2), and the other Fe 2 -metalated ARO proteins characterized herein (Figure S1, PDB entries 8SM7, 8SM8, and 8SM9).…”

“…Recently, we demonstrated that AibH1H2 is a Mn/Fe-dependent monooxygenase that hydroxylates an aliphatic C–H bond of 2-aminoisobutyric acid . The protein components of AibH1H2 stem from a largely uncharacterized structural superfamily, PF04909, containing over 100,000 unique proteins that can individually function as either carboxylases, hydratases, hydrolases, or monooxygenases. − Representatives of the latter enzyme classcollectively defined here as “amidohydrolase-related dinuclear oxygenases” (AROs)include PtmU3 and AibH2, , which employ dinuclear cofactors with different metal identities.…”

“…Recently, we demonstrated that AibH1H2 is a Mn/Fe-dependent monooxygenase that hydroxylates an aliphatic C–H bond of 2-aminoisobutyric acid . The protein components of AibH1H2 stem from a largely uncharacterized structural superfamily, PF04909, containing over 100,000 unique proteins that can individually function as either carboxylases, hydratases, hydrolases, or monooxygenases. − Representatives of the latter enzyme classcollectively defined here as “amidohydrolase-related dinuclear oxygenases” (AROs)include PtmU3 and AibH2, , which employ dinuclear cofactors with different metal identities. In contrast, the other characterized PF04909 enzymes either lack a cofactor or employ a mononuclear metal site that can be Mn or Zn. , The primary structural features that differentiate mono- versus dinuclear active sites, engender monooxygenase functionality, and control the metalation of AibH2 and PtmU3 are unknown.…”

Accessing a synthetic FeIIIMnIV core to model biological heterobimetallic active sites

“…The hyperfine pattern at g = 2 is indicative of an S = 1/2 spin-coupled system containing an S = 5/2 Fe III ion antiferromagnetically coupled to an S = 2 Mn III ion. 6,17–19,32,33,47 Simultaneous least-squares fitting of the spectra for both frequencies provided the g - and A ( 55 Mn)-tensors for the coupled system (Table 2). A rotation between the g - and A-tensors was required to simulate both the S- and X-band spectra with the same parameter set.…”