Coulometric and Spectroscopic Analysis of the Purified Cytochrome d Complex of Escherichia coli: Evidence for the Identification of “Cytochrome a₁” as Cytochrome b₅₉₅

Coulometric and spectroscopic analyses were performed on the three cytochrome components (cytochrome d, cytochrome b₅₅₈, and the cytochrome previously described as cytochrome a₁) of the purified cytochrome d complex, a terminal oxidase of the Escherichia coli aerobic respiratory chain. On the basis of heme extraction, spectroscopic, and coulometric data, the “cytochrome a₁” component was identified as a b-type cytochrome: cytochrome b₅₉₅. The pyridine hemochromogen technique revealed the presence of two molecules of protoheme IX per cytochrome d complex. This quantity of protoheme IX fully accounted for the sum of the cytochrome b₅₅₈ and cytochrome b₅₉₅ components as determined coulometrically. The renaming of cytochrome a₁ as cytochrome b₅₉₅ was further indicated (1) by the lack of any heme a in the complex and (2) by its resolved reduced-minus-oxidized spectrum. The latter was found to be similar to that of cytochrome c peroxidase, which contains protoheme IX. Coulometric titrations and carbon monoxide binding titrations revealed that there are two molecules of cytochrome d per complex. A convenient measurement of the amount of cytochrome b₅₅₈ was found to be the β-band at 531 nm since cytochrome b₅₅₈ was observed to be the only component of the cytochrome d complex with a peak at this wavelength. By use of this method and the extinction coefficient for the purified cytochrome b₅₅₈, it was estimated that there is one molecule of cytochrome b₅₉₅ and one of cytochrome b₅₅₈ per cytochrome complex.