Fig 5.

Spectral analysis of cytochrome cd₁ from WT, ΔnirS mutant, ΔnirN mutant, and ΔnirN mutant plus pLYJ74. (A) UV-visible absorption spectra of oxidized and sodium dithionite reduced periplasmic fraction from WT cells showing characteristic spectra (black arrow) for cytochrome cd₁. (B) Pyridine ferrohemochrome spectrum of purified holo-cytochrome cd₁ (in 50 mM Tris-HCl [pH 7.5]) from WT cells. A characteristic peak for d₁ heme at 620 nm was present (black arrow). (C) UV-visible absorption spectra of oxidized and sodium dithionite-reduced periplasmic fractions from the ΔnirS mutant for cytochrome cd₁. No peak for d₁ heme was found due to the deletion of nirS. (D) Pyridine ferrohemochrome spectrum of the periplasmic fraction (in 50 mM Tris-HCl [pH 7.5]) from the ΔnirS mutant. To avoid the effects from other heme-containing proteins, the periplasmic fraction was purified by DEAE chromatography before analysis. It lacked the characteristic d₁ heme absorption peak at 620 nm. (E) UV-visible absorption spectra of oxidized and sodium dithionite-reduced periplasmic fraction from the ΔnirN mutant for cytochrome cd₁. The absorption peaks for d₁ heme at 620 nm and 663 nm in a reduced form were missing, and instead a new 650-nm shoulder was observed (black dashed arrow). (F) Pyridine ferrohemochrome spectrum of purified semi-apo cytochrome cd₁ (in 50 mM Tris-HCl, pH 7.5) from the ΔnirN mutant. No characteristic peak for d₁ heme was found. (G) UV-visible absorption spectra of the oxidized and sodium dithionite-reduced periplasmic fractions from the ΔnirN mutant with pLYJ74, which carries nirN. The peaks characteristic for d₁ heme (643 nm in oxidized form and 663 and 620 nm in reduced form) were restored (black arrow). (H) Pyridine ferrohemochrome spectrum of purified holo-cytochrome cd₁ (in 50 mM Tris-HCl [pH 7.5]) from the ΔnirN mutant with pLYJ74 with plasmid-borne nirN. A characteristic peak for d₁ heme was observed (black arrow). The results shown come from one data set representative of two independent experiments.