Extended Data Figure 4: The HODM haem subunit has similar properties to the HODM holoenzyme.

a, UV-visible absorption features of alpha HODM (8.5 μM). Ferric (thick solid line), ferrous (thin solid line) Fe²⁺–O₂ (dotted line) and Fe²⁺–CO (dashed line). The major (Soret) absorption band is centred at 409, 423, 418 and 421 nm, respectively. The α/β bands are magnified in the inset. b, Titration of ferric and ferrous-oxy HODM α subunit with DMA. Absorbance changes associated with the haem Soret peak are plotted as a function of DMA concentration. The data are fitted using a hyperbolic function or the Morrison equation. Top, ferric with K_d = 12.6 ± 0.2 mM. Bottom, ferrous-oxy with K_d = 41.0 ± 5.0 μM (errors bars are s.e.m., n = 3). Insets show UV-vis spectra of the titration of DMA (0–50 mM) against ferric and ferrous-oxy HODM α subunit (7.5/4.8 μM). The UV-vis spectrum of α in buffer A was recorded initially and then after each addition of DMA. The direction of the absorbance changes of the ferric and ferrous-oxy Soret peak on substrate addition is measured from 409/415 nm (thick line) to 416/414 nm (dotted line), respectively. c–e, Spectral properties of HODM haem subunit variants E266Q, W180A and R224A, respectively. Ferric (thick black line), ferric in presence of 1 mM DMA (thick grey line), ferrous (thin black line), ferrous in presence of 1 mM DMA (thin grey line) and Fe²⁺–O₂ (dotted line). The α/β bands are magnified in the inset. While the E226Q variant lacks spectroscopic features associated with DMA binding, the R224A variant appears to bind DMA as a 6th ligand to the haem iron in the ferrous state. Oxygen binding can be detected for all variants.