Enzymatic ω-Oxidation: V. FORMS OF PSEUDOMONAS OLEOVORANS RUBREDOXIN CONTAINING ONE OR TWO IRON ATOMS: STRUCTURE AND FUNCTION IN ω-HYDROXYLATION

Abstract

Rubredoxin, a red nonheme iron protein previously shown to be an essential electron carrier in an enzyme system from Pseudomonas oleovorans which hydroxylates fatty acids and hydrocarbons, has been isolated in large quantities by an improved procedure. The protein is a single polypeptide chain with a molecular weight of 19,000 as determined by sedimentation studies and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. It contains a single methionine residue and 10 cysteine residues and, unlike rubredoxins isolated from anaerobic bacteria, histidine and arginine. The isolated form of this protein, which contains 1 atom of iron and is called (1Fe)-rubredoxin, is readily converted to a form which contains 2 atoms of iron and is called (2Fe)-rubredoxin. The oxidized form of (1Fe)-rubredoxin has absorption maxima at 497, 380, and 280 mµ with an A280:A497 ratio of about 6.3, and (2Fe)-rubredoxin has maxima at 495, 378, and 280 mµ with an A280:A495 ratio of about 3.7. The two forms of rubredoxin have highly similar electron paramagnetic resonance spectra. One electron is accepted per atom of iron by each form of rubredoxin, as determined by anaerobic titrations with dithionite or with DPNH in the presence of a catalytic amount of rubredoxin-DPN reductase. Studies on the sulfhydryl content of apo- , (1Fe)- , and (2Fe)-rubredoxin indicated that each iron atom is coordinated by the sulfur atoms of 4 cysteine residues, as has been shown by others for rubredoxin from anaerobic bacteria. Aporubredoxin in which the sulfhydryl groups were protected by formation of the mercaptoethanol mixed disulfide was cleaved with cyanogen bromide, and the two resulting peptides were isolated. Each peptide was converted by treatment with mercaptoethanol and ferrous ions to an iron-containing form with an absorption spectrum typical of a rubredoxin. Determination of the carboxymethylcysteine content of the peptides formed when (1Fe)-rubredoxin was treated with iodoacetamide and then cleaved with cyanogen bromide established that the iron atom is chelated by the group of cysteine residues located near the COOH-terminal end of the molecule. The iron-containing peptide arising from the COOH-terminal portion of rubredoxin can serve as an electron carrier in hydroxylation, whereas that arising from the NH2-terminal portion can not. This was determined by the rate of hydrocarbon hydroxylation under aerobic conditions in the presence of a reduced pyridine nucleotide, a rubredoxin reductase, and the bacterial ω-hydroxylase. (1Fe)- and (2Fe)-rubredoxin function equally well on a molar basis as electron carriers. In contrast, rubredoxins from anaerobic bacteria (Clostridium pasteurianum and Peptostreptococcus elsdenii), the Chromatium high potential iron protein, and spinach ferredoxin show no significant activity over a wide concentration range. Since the immediate environments of the iron atoms in the various rubredoxins are highly similar, it is apparent that other structural features must be essential for the protein to function as an electron carrier in hydroxylation.