Involvement of Cytochrome a in Iron Oxidation of a Moderately Thermophilic Iron-Oxidizing Bacterium, Strain TI-1

The iron-oxidizing activity of a moderately thermophilic iron-oxidizing bacterium, strain TI-1, was located in the plasma membrane. When the strain was grown in Fe²⁺ (60 mM)-salts medium containing yeast extract (0.03%), the plasma membrane had iron-oxidizing activity of 0.129 μmol O₂ uptake/mg/min. Iron oxidase was solubilized from the plasma membrane with 1.0% n-octyl-β-D-glucopyranoside (OGL) containing 25% (v/v) glycerol (pH 3.0) and purified 37-fold by a SP Sepharose FF column chromatography. Iron oxidase solubilized from the plasma membrane was stable at pH 3.0, but quite unstable in the buffer with the pH above 6.0 or below 1.0. The optimum pH and temperature for iron oxidation were 3.0 and 55°C, respectively. Solubilized enzyme from the membrane showed absorption peaks characteristic of cytochromes a and b. Cyanide and azide, inhibitors of cytochrome c oxidase, completely inhibited iron-oxidizing activity at 100 μM, but antimycin A, 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO) and myxothiazol, inhibitors of electron transport systems involved with cytochrome b, did not inhibit enzyme activity at 10 μM. The absorption spectrum of the most active enzyme fraction from SP Sepharose FF column chromatography (4.76 μmol O₂ uptake/mg/min) compared with lower active fractions from the chromatography (0.009 and 2.10 μmol O₂ uptake/mg/min) showed a large α-peak of cytochrome a at 602 nm and a smaller α-peak of cytochrome b at 560 nm. The absorption spectrum of pyridine ferrohemochrome prepared from the most highly purified enzyme showed an α-peak characteristic of heme a at 587 nm, but not the α-peak characteristic of heme c at 550 nm. The cytochrome a, but not cytochrome b, in the most highly purified enzyme fraction was reduced by the addition of ferrous iron at pH 3.0, indicating that electrons from Fe²⁺ were transported to cytochrome a, but not cytochrome b. These results strongly suggest that cytochrome a, but not cytochromes b and c, is involved in iron oxidation of strain TI-1.