Catalytic iron-carbene intermediate revealed in a cytochrome c carbene transferase

Abstract

Recently, heme proteins have been discovered and engineered by directed evolution to catalyze chemical transformations that are biochemically unprecedented. Many of these nonnatural enzyme-catalyzed reactions are assumed to proceed through a catalytic iron porphyrin carbene (IPC) intermediate, although this intermediate has never been observed in a protein. Using crystallographic, spectroscopic, and computational methods, we have captured and studied a catalytic IPC intermediate in the active site of an enzyme derived from thermostable () cytochrome High-resolution crystal structures and computational methods reveal how directed evolution created an active site for carbene transfer in an electron transfer protein and how the laboratory-evolved enzyme achieves perfect carbene transfer stereoselectivity by holding the catalytic IPC in a single orientation. We also discovered that the IPC in cytochrome has a singlet ground electronic state and that the protein environment uses geometrical constraints and noncovalent interactions to influence different IPC electronic states. This information helps us to understand the impressive reactivity and selectivity of carbene transfer enzymes and offers insights that will guide and inspire future engineering efforts.

Ade novoperoxidase is also a promiscuous yet stereoselective carbene transferase

“…Given these data, we assign the new spectroscopically distinct species we describe above to the C45 and Rma-TDE metallocarbenoid intermediates. It should be noted that at this time we cannot definitively assign these spectra as either the non-bridging metallocarbenoid species observed by Lewis et al 45 or the porphyrin-bridging species observed by Hayashi et al 48 in the crystal structures of engineered cytochrome c or N-methylhistidine-ligated myoglobin variant (Mb(H64V,V68A)) respectively. However, given the identical nature of the C45 and Rma-TDE spectra, it would seem likely that the spectra obtained represent the non-bridging ligation observed in the Rma-TDE Me-EDA crystal structure 45 .…”

“…It should be noted that at this time we cannot definitively assign these spectra as either the non-bridging metallocarbenoid species observed by Lewis et al 45 or the porphyrin-bridging species observed by Hayashi et al 48 in the crystal structures of engineered cytochrome c or N-methylhistidine-ligated myoglobin variant (Mb(H64V,V68A)) respectively. However, given the identical nature of the C45 and Rma-TDE spectra, it would seem likely that the spectra obtained represent the non-bridging ligation observed in the Rma-TDE Me-EDA crystal structure 45 . Additionally, while the rate of metallocarbenoid intermediate and subsequent product formation appear relatively low in our stopped-flow experiments, it is worth noting that the selected conditions were necessary for maximizing the quantity of intermediate in the stopped-flow apparatus, and that subsequent activity assays were carried out at higher substrate concentrations (both EDA and styrene), higher temperature and lower ethanol concentrations.…”

“…1c), the latter with an established carbene transferase activity with respect to silanes 45 . Near identical spectra to the putative metallocarbenoid C45 were obtained, though significant differences were apparent between our spectra and those previously reported for the metallocarbenoid complex of Rma-TDE 45 . Since our spectra were collected under more rigorously anaerobic conditions than the previous study (stoppedflow spectrophotometer housed in an anaerobic glovebox; < 5 ppm O2) and at lower temperature, we postulate that the previously reported UV/visible spectra instead represent an alternative, yet currently unidentified, species.…”

“…Mass spectra of the EDA-, BnDA-and t BuDA-generated metallocarbenoids of C45 exhibited the mass differences expected for the adducts, further confirming the nature of the species generated under these conditions. Under identical conditions to C45, we individually mixed ferrous microperoxidase-11 and an engineered cytochrome c from Rhodothermus marinus 45 (Rma-TDE) with EDA ( Fig. 1c), the latter with an established carbene transferase activity with respect to silanes 45 .…”

“…Under identical conditions to C45, we individually mixed ferrous microperoxidase-11 and an engineered cytochrome c from Rhodothermus marinus 45 (Rma-TDE) with EDA ( Fig. 1c), the latter with an established carbene transferase activity with respect to silanes 45 . Near identical spectra to the putative metallocarbenoid C45 were obtained, though significant differences were apparent between our spectra and those previously reported for the metallocarbenoid complex of Rma-TDE 45 .…”