Fig 3.

EhFdp1 is an oxygen reductase. Oxygen consumption was assayed by spectrophotometric (NADH consumption) and amperometric (O₂ consumption determined with a Clark-type oxygen electrode) measurements. Assays were done at room temperature in 50 mM Tris-HCl–18% glycerol (pH 7.5). Electron delivery to EhFDP1 was achieved by assembling a hybrid ET chain composed of NADH, EcRdR, and EcRd. (A) Spectrophotometric NADH consumption was assayed with a reaction mixture volume of 1 ml and sequential addition of 160 μM NADH, 0.17 μM EcRdR, 2.65 μM EcRd, and 36 nM EhFdp1. The decrease in A₃₄₀ shows NADH oxidation at the expense of oxygen reduction mediated by the ET chain with EhFdp1 as the terminal oxygen reductase. (B) Oxygen consumption was assayed with a reaction mixture volume of 1.5 ml and the sequential addition of 1 mM NADH, 0.2 μM EcRdR, 2 μM EcRd, and 150 nM EhFdp1. Oxygen depletion upon the addition of substoichiometric EhFdp1 is shown; addition of catalase (Kat) yielded no production of oxygen, suggesting that EhFdp1 reduces O₂ directly to water.