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.