Directed evolution of cytochrome c for carbon-silicon bond formation: Bringing silicon to life

Abstract

Enzymes that catalyze carbon-silicon bond formation are unknown in nature, despite the natural abundance of both elements. Such enzymes would expand the catalytic repertoire of biology, enabling living systems to access chemical space previously only open to synthetic chemistry. We have discovered that heme proteins catalyze the formation of organosilicon compounds under physiological conditions via carbene insertion into silicon-hydrogen bonds. The reaction proceeds both in vitro and in vivo, accommodating a broad range of substrates with high chemo- and enantioselectivity. Using directed evolution, we enhanced the catalytic function of cytochrome c from Rhodothermus marinus to achieve more than 15-fold higher turnover than state-of-the-art synthetic catalysts. This carbon-silicon bond-forming biocatalyst offers an environmentally friendly and highly efficient route to producing enantiopure organosilicon molecules.

Fig. 1. Heme protein-catalyzed carbon-silicon bond formation

(A) Carbon–silicon bond formation catalyzed by heme and purified heme proteins. (B) Surface representation of the heme-binding pocket of wild-type Rma cyt c (PDB ID: 3CP5). (C) “Active site” structure of wild-type Rma cyt c showing a covalently bound heme cofactor ligated by axial ligands H49 and M100. Amino acid residues M100, V75 and M103 residing close to the heme iron were subjected to site-saturation mutagenesis. (D) Directed evolution of Rma cyt c for carbon–silicon bond formation (reaction shown in (A)). Experiments were performed using lysates of E. coli expressing Rma cyt c variant (OD₆₀₀ = 15; heat-treated at 75 °C for 10 min), 10 mM silane, 10 mM diazo ester, 10 mM Na₂S₂O₄, 5 vol% MeCN, M9-N buffer (pH 7.4) at room temperature under anaerobic conditions for 1.5 h. Reactions performed in triplicate. (E) Carbon–silicon bond forming rates over four generations of Rma cyt c.

Fig. 2. Scope of Rma cyt c V75T M100D M103E-catalyzed carbon–silicon bond formation

Standard reaction conditions: lysate of E. coli expressing Rma cyt c V75T M100D M103E (OD₆₀₀ = 1.5; heat-treated at 75 °C for 10 min), 20 mM silane, 10 mM diazo ester, 10 mM Na₂S₂O₄, 5 vol% MeCN, M9-N buffer (pH 7.4) at room temperature under anaerobic conditions. Reactions performed in triplicate. [a] OD₆₀₀ = 5 lysate. [b] OD₆₀₀ = 0.5 lysate. [c] OD₆₀₀ = 15 lysate. [d] 10 mM silane. [e] OD₆₀₀ = 0.15 lysate.

Fig. 3. Chemoselectivity and in vivo activity of evolved Rma cyt c

(A) Chemoselectivity for carbene Si–H insertion over N–H insertion increased dramatically during directed evolution of Rma cyt c. Standard reaction conditions as described in Fig. 2. Reactions performed in duplicate using heat-treated lysates of E. coli expressing Rma cyt c with protein concentration normalized across variants. Product distribution was quantified after 2 h reaction time (before complete conversion, no double insertion product was observed under these conditions). (B) In vivo synthesis of organosilicon compound 22.