Characterization of DorC from Rhodobacter capsulatus, a c-type cytochrome involved in electron transfer to dimethyl sulfoxide reductase.

The dorC gene of the dimethyl sulfoxide respiratory (dor) operon of Rhodobacter capsulatus encodes a pentaheme c-type cytochrome that is involved in electron transfer from ubiquinol to periplasmic dimethyl sulfoxide reductase. DorC was expressed as a C-terminal fusion to an 8-amino acid FLAG epitope and was purified from detergent-solubilized membranes by ion exchange chromatography and immunoaffinity chromatography. The DorC protein had a subunit Mr = 46,000, and pyridine hemochrome analysis indicated that it contained 5 mol heme c/mol DorC polypeptide, as predicted from the derived amino acid sequence of the dorC gene. The reduced form of DorC exhibited visible absorption maxima at 551.5 nm (alpha-band), 522 nm (beta-band), and 419 nm (Soret band). Redox potentiometry of the heme centers of DorC identified five components (n = 1) with midpoint potentials of -34, -128, -184, -185, and -276 mV. Despite the low redox potentials of the heme centers, DorC was reduced by duroquinol and was oxidized by dimethyl sulfoxide reductase.

On the role of high-potential iron-sulfur proteins and cytochromes in the respiratory chain of two facultative phototrophs

The capability of high potential iron-sulfur proteins (HiPIPs) and soluble cytochromes to shuttle electrons between the bc1 complex and the terminal oxidase in aerobically grown cells of Rhodoferax fermentans and Rhodospirillum salinarum, two facultative phototrophs, was evaluated. In Rs. salinarum, HiPIP and a c-type cytochrome (alpha-band at 550 nm, Em,7=+290 mV) are both involved in the electron transfer step from the bc1 complex to the terminal oxidase. Kinetic studies indicate that cytochrome c550 is more efficient than HiPIP in oxidizing the bc1 complex, and that HiPIP is a more efficient reductant of the terminal oxidase as compared to cytochrome c550. Rs. salinarum cells contain an additional c-type cytochrome (asymmetric alpha-band at 556 nm, Em,7=+180 mV) which is able to reduce the terminal oxidase, but unable to oxidize the bc1 complex. c-type cytochromes could not be isolated from Rf. fermentans, in which HiPIP, the most abundant soluble electron carrier, is reduced by the bc1 complex (zero-order kinetics) and oxidized by the terminal oxidase (first-order kinetics), respectively. These data, taken together, indicate for the first time that HiPIPs play a significant role in bacterial respiratory electron transfer.

The respiratory chain of the halophilic anoxygenic purple bacterium Rhodospirillum sodomense.

The halophilic purple nonsulfur bacterium Rhodospirillum sodomense has been previously described as an obligate phototroph that requires yeast extract and a limited number of organic compounds for photoheterotrophic growth. In this work, we report on chemoheterotrophic growth of R. sodomense in media containing either acetate or succinate supplemented with 0.3-0.5% yeast extract. Plasma membranes isolated from cells grown aerobically in the dark contained three b-type and three c-type membrane-bound cytochromes with Em,7 of +171 +/- 10, +62 +/- 10 and -45 +/- 13 mV (561-575 nm), and +268 +/- 6, +137 +/- 10 and -43 +/- 12 mV (551-540 nm). A small amount of a soluble c-type cytochrome with a mol. mass of 15 kDa (Em, 7 >/= +150 mV) was identified. Spectroscopic and immunological methods excluded the presence of cytochrome of the c2 class and high-potential iron-sulfur proteins. Inhibitory studies indicated that only 60-70% of the respiratory activity was blocked by low concentrations of cyanide, antimycin A, and myxothiazol (10, 0.1, and 0.2 microM, respectively). These results were interpreted to show that the oxidative electron transport chain of R. sodomense is branched, leads to a quinol oxidase that is fully blocked by 1 mM cyanide and that is involved in light-dependent oxygen reduction, and leads to a cytochrome c oxidase that is inhibited by 10 microM cyanide. These features taken together suggest that R. sodomense differs from the closely related species Rhodospirillum salinarum and from other species of the genus Rhodospirillum in that it contains multiple membrane-bound cytochromes c.

Dissecting the Diphenylene Iodonium-Sensitive NAD(P)H:Quinone Oxidoreductase of Zucchini Plasma Membrane.

Quinone oxidoreductase activities dependent on pyridine nucleotides are associated with the plasma membrane (PM) in zucchini (Cucurbita pepo L.) hypocotyls. In the presence of NADPH, lipophilic ubiquinone homologs with up to three isoprenoid units were reduced by intact PM vesicles with a Km of 2 to 7 [mu]M. Affinities for both NADPH and NADH were similar (Km of 62 and 51 [mu]M, respectively). Two NAD(P)H:quinone oxidoreductase forms were identified. The first, labeled as peak I in gel-filtration experiments, behaves as an intrinsic membrane complex of about 300 kD, it slightly prefers NADH over NADPH, it is markedly sensitive to the inhibitor diphenylene iodonium, and it is active with lipophilic quinones. The second form (peak II) is an NADPH-preferring oxidoreductase of about 90 kD, weakly bound to the PM. Peak II is diphenylene iodonium-insensitive and resembles, in many properties, the soluble NAD(P)H:quinone oxidoreductase that is also present in the same tissue. Following purification of peak I, however, the latter gave rise to a quinone oxidoreductase of the soluble type (peak II), based on substrate and inhibitor specificities and chromatographic and electrophoretic evidence. It is proposed that a redox protein of the same class as the soluble NAD(P)H:quinone oxidoreductase (F. Sparla, G. Tedeschi, and P. Trost [1996] Plant Physiol. 112:249-258) is a component of the diphenylene iodonium-sensitive PM complex capable of reducing lipophilic quinones.

Purification and properties of NAD(P)H: (quinone-acceptor) oxidoreductase of sugarbeet cells.

NAD(P)H:(quinone-acceptor) oxidoreductase [NAD(P)H-QR], a plant cytosolic protein, was purified from cultured sugarbeet cells by a combination of ammonium sulfate fractionation, FPLC Superdex 200 gel filtration, Q-Sepharose anion-exchange chromatography, and a final Blue Sepharose CL-6B affinity chromatography with an NADPH gradient. The subunit molecular mass is 24 kDa and the active protein (94 kDa) is a tetramer. The isoelectric point is 4.9. The enzyme was characterized by ping-pong kinetics and extremely elevated catalytic capacity. It prefers NADPH over NADH as electron donor (kcat/Km ratios of 1.7 x 10(8) M-1 S-1 and 8.3 x 10(7) M-1 S-1 for NADPH and NADH, respectively, with benzoquinone as electron acceptor). The acridone derivative 7-iodo-acridone-4-carboxylic acid is an efficient inhibitor (I0.5 = 5 x 10(-5) M), dicumarol is weakly inhibitory. The best acceptor substances are hydrophilic, short-chain quinones such as ubiquinone-0 (Q-0), benzoquinone and menadione, followed by duroquinone and ferricyanide, whereas hydrophobic quinones, cytochrome c and oxygen are reduced at negligible rates at best. Quinone acceptors are reduced by a two-electron reaction with no apparent release of free semiquinonic intermediates. This and the above properties suggest some relationship of NAD(P)H-QR to DT-diaphorase, an animal flavoprotein which, however, has distinct structural properties and is strongly inhibited by dicumarol. It is proposed that NAD(P)H-QR by scavenging unreduced quinones and making them prone to conjugation may act in plant tissues as a functional equivalent of DT-diaphorase.