Figure 1. Reaction of sMMO with O₂.

a, The catalytic cycle includes stable MMOH^ox and MMOH^red and detectable transient species O, P*, P, Q and T; transient states QS and R were predicted from kinetic, spectroscopic, and chemical studies. b, An in situ transient difference electronic absorption spectrum of the reaction mixture (vs anaerobic MMOH^red) in the TR³ instrument at Δt ≈ 3.0 s (red, 1.0 × 0.1² mm probe volume) in comparison with spectrum of Q reconstructed from stopped-flow kinetic traces (markers, blue). c, Transient ¹⁶O₂ – ¹⁸O₂ difference resonance Raman spectra of sMMO reveal vibrations of iron-bound oxygen atoms. Measurement conditions: pH 7.0, 4 °C, Δt ≈ 3.0 s. Spectra recorded without the substrate (i) show marked changes when 0.45 mM CH₄ (ii), 3.5 mM furan (iii) or 0.45 mM CD₄ (iv) is added. d, Solvent vibrations in the absolute resonance Raman spectra are sensitive to H₂O (i)/D₂O (ii) substitution. ¹⁶O₂ – ¹⁸O₂ difference spectra of sMMO recorded in H₂O (iii) show little sensitivity to D₂O (iv) substitution. The upshift of ¹⁸O vibration of Q and the appearance of a low-frequency shoulder (marked with S) in D₂O is attributed to Fermi resonance with a protein-derived metal ligand.