Febs Letters Volume 109, Number 1-2./Volume 110, Number 1-2. [antikvár]

Volume 109, number 2 FEBS LETTERS January 1980 THE PURIFICATION OF A RESPIRATORY OXIDASE COMPLEX FROM ESCHERICHIA COLI Graeme A. REID* and W. John INGLEDEW+ Department of Biochemistry, Medical Sciences Institute, University of Dundee, Dundee, DDI 4HN and '''Department of Biochemistry and Microbiology, University of St Andrews, St Andrews, Scotland Received 17 October 1979 I. Introduction Escherichia coli has the abihty to regulate the composition of its respiratory chain according to environmental factors [1,2]. When grown aerobically beyond exponential phase, cells produce two respiratory oxidases, cytochrome o and cytochrome rf [3]. When grown anaerobically with a non-fermentable carbon source (e.g., glycerol) plus fumarate, cells contain fumarate reductase as the major terminal respiratory enzyme. However these cells also contain very high concentrations of the oxidase cytochrome d, while no cytochrome o is spectroscopically detectable. Cytochromes ?555, Ös58 and fli, are found in addition to cytochrome d [2]. Cytochrome rf is a 1 electron acceptor, but the reduction of O2 to HjO requires the transfer of 4 electrons. Known enzymes which also catalyse this oxidase reaction have been shown to contain 4 redox centres: mitochondrial cytochrome c oxidase has two haem a groups plus 2 redox-active Cu atoms [4] while Pseudomonas cytochrome oxidase (nitrite reductase) contains 2 haem c plus 2 haem di [5] moieties. We have solubilized and purified cytochrome d to investigate the nature and number of redox centres present and find that cytochromes b^ and 6558 copurify with cytochrome d. Evidence from gel electrophoresis suggests that the oxidase is a protein complex containing both cytochromes b and d. 2. Materials and methods 2.\. Growth of cells Escherichia coli strain EMG-2 (prototroph) was grown in 20 1 batches on the mineral salts medium (CR medium) [6] supplemented with vitamin-free casamino acids (0.1%, w/v), glycerol (0.5%, w/v) and fumarate (50 mM). The autoclaved medium was briefly bubbled with nitrogen gas and the cuhure vessel sealed with a rubber stopper to maintain anae-robiosis. An inoculum of 450 ml was used for each 20 1 batch. 2.2. Harvesting arui cell breakage Cells were harvested in late exponential phase in an MSE continuous action rotor (43118-503) at 16 000 rev./min with ~300 ml/min flow rate. The pellet was resuspended in CR medium and centrifuged at 10 000 X g for 15 min at 4°C. This washing procedure was repeated once. Cells were broken in the French pressure cell and electron transport particles were prepared as in [2]. 2.3. Optical spectroscopy Difference spectra were measured with the split-beam spectrophotometer [7] either at room temperature or at 77 K. The molar extinction coefficient of cytochrome b in solubilized preparations was determined by comparing reduced minus oxidised difference spectra (room temperature) with alkaline pyridine haemochromogen spectra. Thus ^s60 -576 was calculated to be 12 mM"'. 2.4. Polyacrylamide gels Electrophoresis under non-dissociating conditions was performed, at pH 8.9, in 7.3% (w/v) polyacrylamide gels as in [8]. Gels were stained for iron as in [9] and for haem as follows: ethanolic o-toUdine was acidified with glacial acetic acid to 10% (v/v). Gels were immersed in this reagent 2 min after which the excess toUdine reagent was removed. HjOi 3% (v/v) was added to develop the transient blue stain. ElsevierlNorth-Holland Biomedical Press 1980