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 by the lack of any heme a in the complex and 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 beta-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.
D002248:Carbon Monoxide / Q000378:metabolism; D003573:Cytochrome b Group; D017937:Cytochrome d Group; D003580:Cytochromes / Q000302:isolation & purification* / Q000378:metabolism; D045263:Cytochromes a1; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D011199:Potentiometry; D011485:Protein Binding; D013053:Spectrophotometry
D003573:Cytochrome b Group; D003580:Cytochromes; D029968:Escherichia coli Proteins; C472600:cytochrome b595, E coli; D002248:Carbon Monoxide; D045263:Cytochromes a1; D017937:Cytochrome d Group