A component of the NADH-cytochrome o reductase system of Vitreoscilla purified to near homogeneity as judged by disc electrophoresis had a molecular weight of 61,000 when estimated by gel filtration. Electrophoresis on polyacrylamide gels containing sodium dode-cy1 sulfate showed that the enzyme consists of two 35,000-dalton subunits. The enzyme contains one FAD and two non-heme irons per molecule all noncovalently bound. Acid-labile sulfide was not detectable. The flavoprotein can catalyze the

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... reduction by NADH of partially purified cytochrome o and a number of artificial electron acceptors such as ferricyanide, cytochrome 0, dichlorophenolindophenol, and p-iodonitrotetrazolium violet, but not pure cytochrome 0. The reduction of partially purified cytochrome o was assayed aerobically by following formation of the oxygenated cytochrome; the kinetics of this reduction were hyperbolic with respect to NADH. The anaerobic reduction of cytochrome o catalyzed by the reductase in the presence of excess NADH occurred in two phases: an initial rapid decrease in absorption of the oxidized cytochrome at 405 and 540 nm followed by a slow shift of these maxima to 423 and 555 nm, respectively. This result is evidence for a sequential reduction of the high and low potential hemes of cytochrome 0. The reductase can accept 4 electrons as determined by anaerobic titration of the fully reduced enzyme with ferricyanide and photochemical titration in the presence of safranin 0. Midpoint potentials of the four oxidation-reduction centers estimated from the latter titration were in the range of -257 to -318 mV. The flavosemiquinone was present during intermediate stages of the titrations. Titration of the oxidized protein with NADH reduced only two of the four electron centers. The inability of NADH to fully reduce the purified reductase and the unreactivity of the enzyme with pure cytochrome o suggest that other components are required for the reduction of the cytochrome. Cytochrome o is a common terminal oxidase in bacteria. The respiratory chain of Vitreoscilla, a filamentous, strictly aerobic prokaryote, is unique in that it does not contain c-, a-, or d-type cytochrome and employs cytochrome o as its only terminal oxidase (1). This cytochrome has been purified to homogeneity and its properties have been studied in detail (2-4). It was reported previously that a NADH-cytochrome o reductase co-purified with the cytochrome and that oxygen uptake with NADH as substrate was primarily associated with those preparations containing high NADH-cytochrome o reductase activity (5). Subsequently, evidence for the involvement of a flavoprotein in the NADH-cytochrome o oxidase reaction was obtained (6). This paper reports the purification of NADH-cytochrome o reductase, some of its electronaccepting and -transferring properties, and discusses some implications of these properties with regard to the mechanism of reduction of cytochrome 0. METHODS Purification of NADH-Cytochrome o Reductase-Culture of cells, preparation of cell-free extracts, removal of nucleic acids with protamine sulfate, and ammonium sulfate fractionation were performed as previously described (7) . Subsequent steps, herein described, were carried out at 0-4'C. The fraction precipitated a t 45 to 65% ammonium sulfate saturation was dissolved in and dialyzed against 0.02 M sodium phosphate buffer, pH 7.2, and adsorbed onto a DEAE-cellulose column (4.7 X 55 cm) equilibrated with the same buffer. The column was eluted with 3 liters of a linear gradient of 0 to 0.5 M sodium chloride in equilibration buffer. The reductase eluted a t 0.3 to 0.4 M sodium chloride and was separated from the cytochrome which eluted at 0.2 to 0.3 M (Fig. 1) . The pooled cytochrome o fractions were concentrated and chromatographed on Sephadex G-75 according to Tyree and Webster (2). The resulting cytochrome preparation was used for NADH-cytochrome o reductase assays. It had an A4~"/A~n" of approximately 1.8, indicating a purity of about 75%. Fractions containing NADH-cytochrome o reductase activity were pooled, concentrated by ultrafiltration using an Amicon PMlO ultrafilter, and chromatographed on Sephadex G-200 (5 X 84 cm) equilibrated with 0.02 M sodium phosphate buffer, pH 7.5, containing 0.1 M sodium chloride. Gel filtration was repeated on a Sephadex G-100 column (4.7 X 50 cm) under the same conditions. The pooled reductase fractions were then concentrated and further purified by preparative electrophoresis on 10% polyacryIamide gel slabs (3 X 140 X 170 mm) according to Davis (8). Gel slabs were made in a Desaga polymerizing chamber and consisted of 50 ml of separation gel, 6 ml of stacking gel, and 4 ml of sample gel containing 40 to 50 mg of protein. Electrophoresis was run for 6 to 7 h a t a constant current of 30 mA in a Desaga TLE chamber with a Brinkmann-regulated power supply. Temperature was maintained a t 0-4°C with a Haake circulating pump. The bright yellow band that migrated toward the anode stained for NADH dehydrogenase activity using p-iodonitrotetrazolium violet (9). This band was sliced off the gel slab and extracted with three 20-ml washes of 0.02 M sodium phosphate buffer, pH 7.5, containing 1.0 M sodium chloride, each wash being allowed to equilibrate a t 4°C for 12 to 16 h. The combined washes were dialyzed against 0.02 M sodium phosphate buffer, pH 7.5, concentrated with Aquacide I1 A (Calbiochem) and stored frozen as 0.5-ml aliquots. Purity of the preparation was determined by disc electrophoresis (8). Determination of Molecular Weight-Molecular weight was estimated by gel filtration on Sephadex G-100 (1.9 X 115 cm) equilibrated with 0.02 M sodium phosphate buffer, pH 7.5, containing 0.