@article{mbs:/content/journal/micro/10.1099/00221287-132-6-1525, author = "Poole, Robert K. and Baines, Baldev S. and Appleby, Cyril A.", title = "Haemoprotein b-590 (Escherichia coli), a Reducible Catalase and Peroxidase: Evidence for its Close Relationship to Hydroperoxidase I and a ‘Cytochrome a1b’ Preparation", journal= "Microbiology", year = "1986", volume = "132", number = "6", pages = "1525-1539", doi = "https://doi.org/10.1099/00221287-132-6-1525", url = "https://www.microbiologyresearch.org/content/journal/micro/10.1099/00221287-132-6-1525", publisher = "Microbiology Society", issn = "1465-2080", type = "Journal Article", abstract = "A reducible hydroperoxidase, haemoprotein b-590, has been purified 16-fold from a soluble fraction of Escherichia coli K12, grown anaerobically with glycerol and fumarate. The M r of the native protein, determined by gel filtration, was 331000 although a minor, smaller species with a M r of 188000 was also detected; both had catalase activities. Based on the subunit M r, determined from SDS gel electrophoresis to be 75000, the above species are tentatively identified as tetramers and dimers, respectively. The isoelectric point of both species was 4·4. The absorption spectrum of the isolated haemoprotein is typical of ferric, high-spin haem. The A 405/A 280 ratio never exceeded 0·27, a value half of that obtained for E. coli hydroperoxidase I. On reduction with dithionite, the ?, ?, and ? bands were at 441, 559 and 590 nm respectively, the ?-band being unusually distinct. Treatment of the reduced form with CO gave a sharp prominent ?-band at 426 nm and caused significant shifts of the ? and ? bands to shorter (574 and 545 nm) wavelengths. The pyridine haemochrome spectra showed the haem to be protohaem IX; the spectra were featureless between 580 and 630 nm, thus excluding the presence of haem a. However, some features of the difference spectra of the haemoprotein were reminiscent of cytochrome a 1, notably the maxima in reduced minus oxidized spectra at 444 and 593 nm and the peaks and troughs in CO difference spectra at 426 and 446 nm respectively. The haemoprotein had high catalase activity: V max was 2·3 × 106 mol H2O2 (mol haem)?1 min?1 and the K m was 11 mM. At 10 mM-H2O2 the first order rate constant was 0·3 × 107 M?1 s?1. The haemoprotein was also a peroxidase with o-dianisidine or 2,3’,6-trichloroindophenol as substrates; for the latter substrate, the K m was 0·18 mM. It is concluded that haemoprotein b-590 strongly resembles the hydroperoxidase I purified by Claiborne & Fridovich (Journal of Biological Chemistry 254, 4245-4252, 1979) and that a similar haemoprotein was mistaken for a cytochrome a 1 b complex by Barrett & Sinclair (Abstracts of the 7th International Congress of Biochemistry, Tokyo, H-107, p. 907, 1967).", }