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

Abstract

The e.p.r. signals attributable to a cytochrome bd-type ubiquinol:O2 oxidoreductase (cytochrome b-558-b-595-d) were studied in a cytoplasmic membrane preparation of Escherichia coli that had been grown on glycerol with fumarate as respiratory-chain oxidant. Two major high-spin ferric haem signals were resolved on the basis of their potentiometric behaviour: a rhombic high-spin species (gx = 6.25, gy = 5.54) was assigned to haem b-595, and an axial high-spin (gx = 5.97, gy = 5.96) species was assigned to the haem d. These signals titrated with Em.7 values of 154 and 261 mV respectively, corresponding closely to optically determined values for haem b-595 and haem d. At high potentials (greater than 300 mV) the rhombic species attributable to haem b-595 underwent a partial transition to a second rhombic species with g-values of 6.24 (gx) and 5.67 (gy). The high-spin ferric haem spectra were affected by O2, CO, cyanide and pH. A low-spin ferric haem signal was observed at g = 3.3 (gz), which titrated with an Em.7 of 226 mV, and this was assigned to haem b-558. The data support a model for cytochrome bd with two ligand-binding sites, a single haem d and a single haem b-595.

Cyanide-binding Site of bd-type Ubiquinol Oxidase from Escherichia coli

“…The line shapes of both the rhombic and axial components are altered, with the axial component being slightly broader and the rhombic signal being further distorted. These observations are essentially consistent with those reported for the membrane-bound bd-type ubiquinol oxidase (32). However, the intensity of the high spin signals did not change appreciably compared with that of the air-oxidized O 2 -bound state before addition of cyanide.…”

Cytochrome d axial ligand of the bd‐type terminal quinol oxidase from Escherichia coli

“…The minor ferric low-spin signal observed in [lo] could be seen at g = 2.46 (g,), 2.32 (g,), and 1.83 (g,). The other ferric low-spin signal also could be observed at g = 3.31 at 5K as previously reported [13,14], but not at 15K.…”

Orientation of the haems of the ubiquinol oxidase: O2 reductase, cytochrome bo of Escherichia coli

“…They did not assign this high-spin haem; our results eliminate the possibility that it arises from the haem o. The high-spin EPR signals of the cytochrome bo are much weaker in comparison to the low-spin component than the high-spin signals of the cytochrome bd [7,16,171. This is only partly due to the number of high-spin haems in each system (two for 'bd', one for '0') and the somewhat greater transition probability of the low-spin b associated with cytochrome bo compared with the low-spin cytochrome b of the bd; coupling to a copper(I1) atom is involved in the cytochrome bo as well [7].…”