Fig. 1.

(A) Overall reaction of carbene transfer to an alkyne catalyzed by an engineered hemeprotein. (B) Proposed catalytic cycle of carbene transfer to phenylacetylene to form cyclopropene and bicyclobutane structures. (C) Screening of hemin and hemeprotein catalysts for bicyclobutane formation (BSA = bovine serum albumin; for sources, sequences, and mutations in Bacillus megaterium P411-S1 and other proteins, see Supplementary Materials). (D) X-ray crystal structure of P411-E10 (PDB ID: 5UCW) and view of its distal heme region. The heme axial ligand is S400, and amino acid residues V78, L263 and S438 are shown as gray sticks. (E) Directed evolution of P411-E10 for bicyclobutane formation (using phenylacetylene and EDA as substrates; numbers refer to total turnovers to product (TTN) measured). Experiments were performed on analytical scale using suspensions of E. coli expressing P411-E10 variants (OD600 = 10–30), 10 mM phenylacetylene, 10 mM EDA, 5 vol% EtOH, M9-N buffer (pH 7.4) at room temperature under anaerobic conditions for 6 h. Reactions performed in quadruplicate. Here TTN refers to the total desired product, as quantified by gas chromatography (GC), divided by total hemeprotein. (Note: because bicyclobutane formation requires two carbene transfers, the number of carbene transfers the hemeprotein catalyzes is 2 x TTN in these reactions.) Further details on reaction conditions and data analysis are provided in the Supplementary Materials.