Potentiometric analysis of the cytochromes of an Escherichia coli mutant strain lacking the cytochrome d terminal oxidase complex

Abstract

A combination of potentiometric analysis and electrochemically poised low-temperature difference spectroscopy was used to examine a mutant strain of Escherichia coli that was previously shown by immunological criteria to be lacking the cytochrome d terminal oxidase. It was shown that this strain is missing cytochromes d, a,, and b558 and that the cytochrome composition of the mutant is similar to that of the wild-type strain grown under conditions of high aeration. The data indicate that the high-aeration branch of the respiratory chain contains two cytochrome components, b556 (midpoint potential [Em] = +35 mV) and cytochrome o (Em = +165 mV). The latter component binds to CO and apparently has a reduced-minusoxidized split-a. band with peaks at 555 and 562 nm. When the wild-type strain was grown under conditions of low aeration, the components of the cytochrome d terminal oxidase complex were observed: cytochrome d (Em = +260 mV), cytochrome a, (En = +150 mV) and cytochrome b558 (Em = +180 mV). All cytochromes appeared to undergo simple one-electron oxidation-reduction reactions. In the absence of CO, cytochromes b558 and o have nearly the same Em values. In the presence of CO, the E, of cytochrome o is raised, thus allowing cytochromes b558 and o to be individually quantitated by potentiometric analysis when they are both present.

The cytochromes of anaerobically grown Escherichia coli. An electron-paramagnetic-resonance study of the cytochrome bd complex in situ

“…We have assigned the rhombic high-spin ferric haem signal to haem b-595 on the basis of the similarity of its Em,7 to the published values for this centre (Lorence et al, 1984a), and this assignment is in agreement with that by Hata et al (1987). The optical spectrum of haem b-595 is typical of a high-spin haem b (Lorence et al, 1986), so that the observation of a high-spin e.p.r.…”

“…Our assignment of the axial high-spin ferric haem signal to haem d is based on its potentiometric and ligand-binding behaviour. This signal has an Em,7 of 261 mV, which is raised to above 400 mV in the presence of CO, as has been reported for haem d on the basis of optically monitored redox titrations (Lorence et al, 1984a). The assignment is strengthened by comparison with the e.p.r.…”

“…The continuous line represents an n = I fit of the data to the Nernst equation (see the text). cytochrome bd on the basis of published potentiometric titrations (Lorence et al, 1984a;Reid & Ingledew, 1979). We assign the g = 3.3 signal to haem b-558 (see the Discussion section).…”

“…CO therefore binds to haem d and raises its Em 7 to greater than 400 mV (the highest potential of the titration). The effect of CO on the low-spin haem b-558 signal was not determined, as the Em 7 of this haem is not perturbed by CO in optical redox titrations (Lorence et al, 1984a).…”

“…The components of the complex in membrane preparations from both aerobically and anaerobically grown cells have been resolved potentiometrically by using optical difference spectroscopy. In aerobically grown cells the haem d, haem b-595 and haem b-558 have midpoint potentials (Em,7) of 247, 150 and 182 mV respectively (Lorence et al, 1984a), whereas in anaerobically grown cells these components have midpoint potentials of 280, 150 and 250 mV respectively (Reid & Ingledew, 1979). Although much work has been done on the optical properties of this oxidase, comparatively little is known about its e.p.r.…”