Involvement of cytochrome a in iron oxidation of a moderately thermophiliciron-oxidizing bacterium, strain TI-1

The iron-oxidizing activity of a moderately thermophilic iron-oxidizing bac terium, strain TI-1, was located in the plasma membrane. When the strain wa s grown in Fe2+ (60 mM)-salts medium containing yeast extract (0.03%), the plasma membrane had iron-oxidizing activity of 0.129 mu mol O-2 uptake/mg/m in. Iron oxidase was solubilized from the plasma membrane with 1.0% n-octyl -beta-D-glucopyranoside (OGL) containing 25% (v/v) glycerol (pH 3.0) and pu rified 37-fold by a SP Sepharose FF column chromatography. Iron oxidase sol ubilized 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 temper ature for iron oxidation were 3.0 and 55 degrees C, respectively. Solubiliz ed enzyme from the membrane showed absorption peaks characteristic of cytoc hromes a and b. Cyanide and azide, inhibitors of cytochrome c oxidase, comp letely inhibited iron-oxidizing activity at 100 mu M, but antimycin A, 2-n- heptyl-4-hydroxyquinoline-N-oxide (HOQNO) and myxothiazol, inhibitors of el ectron transport systems involved with cytochrome b, did not inhibit enzyme activity at 10 mu M. The absorption spectrum of the most active enzyme fra ction from SP Sepharose FF column chromatography (4.76 mu mol O-2 uptake/mg /min) compared with lower active fractions from the chromatography (0.009 a nd 2.10 mu mol O-2 uptake/mg/min) showed a large alpha-peak of cytochrome a at 602 nm and a smaller alpha-peak of cytochrome b at 560 nm. The absorpti on spectrum of pyridine ferrohemochrome prepared from the most highly purif ied enzyme showed an alpha-peak characteristic of heme a at 587 nm, but not the alpha-peak characteristic of heme c at 550 nm. The cytochrome a, but n ot cytochrome, in the most highly purified enzyme fraction was reduced by t he addition of ferrous iron at pH 3.0, indicating that electrons from Fe2were transported to cytochrome a, but not cytochrome b. These results stron gly suggest that cytochrome a, but not cytochromes b and c, is involved in iron oxidation of strain TI-1.