Haemoprotein b-590 (Escherichia coli), a Reducible Catalase and Peroxidase: Evidence for its Close Relationship to Hydroperoxidase I and a "Cytochrome a1 b" Preparation

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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 Mr of the native protein determined by gel filtration, was 331,000 although a minor, smaller species with a Mr of 188,000 was also detected; both had catalase activities. Based on the subunit Mr, determined from SDS gel electrophoresis to be 75,000, 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 A405/A280 ratio never exceeded 0.27, a value half of that obtained for E. coli hydroperoxidase I. On reduction with dithionite, the .gamma., .beta., and .alpha. bands were at 441, 559 and 590 nm respectively, the .alpha.-band being unusually distinct. Treatment of the reduced form with CO gave a sharp prominent .gamma.-band at 426 nm and caused significant shifts of the .alpha. and .beta. 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 a1, 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 .times. 106 mol H2O2 (mol haem)-1 min-1 and the Km was 11 nM. At 10 mM-H2O2 the first order rate constant was 0.3 .times. 107 M-1 s-1. The haemoprotein was also a peroxidase with o-dianisidine or 2,3'', 6-trichloroindophenol as substrate; for the latter substrate, the Km was 0.18 mM. It is concluded that haemoprotein b-590 stongly resembles the hydroperoxidase I purified by Claiborne and Fridovich (Journal of Biological Chemistry 254, 4245-4252, 1979) and that a similar haemoprotein was mistaken for a cytochrome a1 b complex by Barrett and Sinclair (Abstracts of the 7th International Congress of Biochemistry, Tokyo, H-107, p. 907, 1967).