1887

Abstract

Summary: A reducible hydroperoxidase, haemoprotein -590, has been purified 16-fold from a soluble fraction of K12, grown anaerobically with glycerol and fumarate. The of the native protein, determined by gel filtration, was 331000 although a minor, smaller species with a of 188000 was also detected; both had catalase activities. Based on the subunit , 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 / ratio never exceeded 0·27, a value half of that obtained for hydroperoxidase I. On reduction with dithionite, the γ, β, and α bands were at 441, 559 and 590 nm respectively, the α-band being unusually distinct. Treatment of the reduced form with CO gave a sharp prominent γ-band at 426 nm and caused significant shifts of the α and β 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 However, some features of the difference spectra of the haemoprotein were reminiscent of cytochrome , notably the maxima in reduced 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: was 2·3 × 10 mol HO (mol haem) min and the was 11 mM. At 10 mM-HO the first order rate constant was 0·3 × 10 M s. The haemoprotein was also a peroxidase with -dianisidine or 2,3”,6-trichloroindophenol as substrates; for the latter substrate, the was 0·18 mM. It is concluded that haemoprotein -590 strongly resembles the hydroperoxidase I purified by Claiborne & Fridovich ( , 4245-4252, 1979) and that a similar haemoprotein was mistaken for a cytochrome complex by Barrett & Sinclair (, Tokyo, H-107, p. 907, 1967).

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/content/journal/micro/10.1099/00221287-132-6-1525
1986-06-01
2019-10-22
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