Minus Oxidized Difference Spectra Research Articles

Bactericidal Effect of 405-nm LED Light against <i>Aggregatibacter Actinomycetemcomitans</i> Is Due Primarily to Disruption of Respiratory Chain Terminal Members Cytochrome <i>bd</i> Oxidase and Quinol Peroxidase

Irradiation with 405-nm visible violet LED light without additional photosensitizers decreased the viability of the aggressive periodontopathic bacterium Aggregatibacter actinomycetemcomitans. The number of CFU/mL decreased linearly on a logarithm chart versus irradiation time, with a 1-log reduction time of 1.32 min. The antimicrobial photodynamic effect of 405-nm LED light involved inhibition of the activity of membrane-bound cytochrome bd, a terminal quinone: oxygen oxidoreductase, and quinol peroxidase, a terminal quinone:H2O2 oxidoreductase. The 405-nm LED irradiation reduced minus oxidized difference spectrum showed that the 640-nm peak (α-peak of heme d) completely disappeared, and the height of the 556-nm (α-peak of hemes b and c) and Soret band (425 nm; γ-peak of hemes b, c, and d) was reduced to approximately half of the peak heights of non-irradiated controls. Survival of bacteria-injected silkworm larvae was also examined. Fifth-instar silkworm larvae were almost completely killed by approximately 40 h after bacterial injection, but almost all silkworm larvae irradiated with 405-nm LED light (20 mW/cm2 for 5 min, energy density: 6 J/cm2) survived, similar to controls not injected with bacteria, indicating that 405-nm LED light killed the injected bacteria. The bactericidal effect of 405-nm blue-light on A. actinomycetemcomitans is primarily due to disruption of cytochrome bd oxidase and quinol peroxidase of the respiratory chain.

The effect of cytochrome level in rats with sepsis on mitochondrial function of hepatocytes

Objective To investigate the effects of cytochrome level and oxidase activity of cytochrome C in rats with sepsis on mitochondrial bioenergy synthesis of hepatocytes and liver functions. Methods 24 SD rats were divided into normal control group (Group C) and sepsis group (Group S), with 12 rats in each group. The sepsis model of the rats was established by the cecal ligation and puncture. After 18 h of moulding, the rats were executed. Their blood was taken to test alanine aminotransferase (ALT), aspartate aminotransferase (AST) and mitochondrial aspartate aminotransferase (m-AST). The morphologies of hepatocytes and mitochondria were observed using light microscope and electron microscope respectively. Using reduced minus oxidized difference spectra, the contents of mitochondrial cytochromes C, C1, B and AA3 and the oxidase activity of cytochrome C were determined. The potential level of mitochondrial membrane was tested. The content of adenosine triphosphate (ATP) in hepatocytes was measured. Results Compared with Group C, the ALT, AST and m-AST in the serum of Group S rose significantly [(233.02±110.16) U/L vs. (29.81±13.16) U/L, (742.56±441.41) U/L vs. (99.78±41.67) U/L and (412.78±252.56) U/L vs. (46.23±24.24) U/L, P= 0.000, 0.001, 0.001, respectively]. The ATP content in hepatocytes dropped significantly [(19.27±8.76) nmol/mg vs. (94.65±17.79) nmol/mg, P=0.000]. Cytochromes c and AA3 in mitochondria declined significantly [(0.10±0.04) nmol/mg vs. (0.17±0.03) nmol/mg and (0.21±0.04) nmol/mg vs. (0.38±0.09) nmol/mg, P=0.000, 0.000]. The activity of cytochrome C oxidase was decreased significantly [(734.14±107.37) nmol/(min·mg prot) vs. (1 279.75±186.77) nmol/(min·mg prot), P=0.000]. The potential level of mitochondrial membrane dropped significantly (1.75±0.96 vs. 3.81±1.16, P=0.000). Conclusion In the case of sepsis, the content of mitochondrial cytochromes in hepatocytes decreases and the oxidase activity of cytochrome C drops. As a result, these leads to mitochondrial dysfunction and acute injury of hepatocytes. Key words: Sepsis; Liver; Mitochondrial; Cytochrome; Cytochrome C oxidase

Quantitation of Cytochromes b559, b6, and f, and the Core Component of Photosystem I P700 in Cyanobacterial Cells

Cytochrome (Cyt) b559, an important and essential core component of photosystem II in the photosynthetic electron transport system, is a heme-bridged heterodimer protein composed of an alpha subunit (PsbE) and a beta subunit (PsbE), and its reduced form has an absorption maximum in the α-band at 559 nm. The amounts of Cyt b559 can be determined by spectrophotometrical measurement of reduced minus oxidized difference spectra that are normalized with absorbance of isosbestic point at 580 nm. The authors use differential extinction coefficients of Cyt b559 [Δε(559-580 nm) = 15.5 mM-1·cm-1], which have been reported by Garewal and Wasserman (1974). In addition to the Cyt b559, this procedure can be used for quantitation of Cyt b6 and Cyt f, the subunits of the Cyt b6/f complex, and P700, one of the core components of photosystem I. This protocol, which is adapted from Fujita and Murakami (1987), is used in a cyanobacterium, Synechococcus elongatus PCC 7942, and also in other cyanobacterial strains including Synechocystis sp. PCC 6803.

Heme/heme redox interaction and resolution of individual optical absorption spectra of the hemes in cytochrome bd from Escherichia coli

Cytochrome bd is a terminal component of the respiratory chain of Escherichia coli catalyzing reduction of molecular oxygen to water. It contains three hemes, b 558, b 595, and d. The detailed spectroelectrochemical redox titration and numerical modeling of the data reveal significant redox interaction between the low-spin heme b 558 and high-spin heme b 595, whereas the interaction between heme d and either hemes b appears to be rather weak. However, the presence of heme d itself decreases much larger interaction between the two hemes b. Fitting the titration data with a model where redox interaction between the hemes is explicitly included makes it possible to extract individual absorption spectra of all hemes. The α- and β-band reduced- minus-oxidized difference spectra agree with the data published earlier ([22] J.G. Koland, M.J. Miller, R.B. Gennis, Potentiometric analysis of the purified cytochrome d terminal oxidase complex from Escherichia coli, Biochemistry 23 (1984) 1051–1056., and [23] R.M. Lorence, J.G. Koland, R.B. Gennis, Coulometric and spectroscopic analysis of the purified cytochrome d complex of Escherichia coli: evidence for the identification of “cytochrome a 1” as cytochrome b 595, Biochemistry 25 (1986) 2314–2321.). The Soret band spectra show λ max = 429.5 nm, λ min ≈ 413 nm (heme b 558), λ max = 439 nm, λ min ≈ 400 ± 1 nm (heme b 595), and λ max = 430 nm, λ min = 405 nm (heme d). The spectral contribution of heme d to the complex Soret band is much smaller than those of either hemes b; the Soret/α (Δ A 430:Δ A 629) ratio for heme d is 1.6.

Functional Characterization of AtATM1, AtATM2, and AtATM3, a Subfamily of Arabidopsis Half-molecule ATP-binding Cassette Transporters Implicated in Iron Homeostasis

The functional capabilities of one of the smallest subfamilies of ATP-binding cassette transporters from Arabidopsis thaliana, the AtATMs, are described. Designated AtATM1, AtAATM2, and AtATM3, these half-molecule ABC proteins are homologous to the yeast mitochondrial membrane protein ATM1 (ScATM1), which is clearly implicated in the export of mitochondrially synthesized iron/sulfur clusters. Yeast ATM1-deficient (atm1) mutants grow very slowly (have a petite phenotype), are respiration-deficient, accumulate toxic levels of iron in their mitochondria, and show enhanced compensatory high affinity iron uptake. Of the three Arabidopsis ATMs, AtATM3 bears the closest functional resemblance to ScATM1. Heterologously expressed AtATM3 is not only able to complement the yeast atm1 petite phenotype but is also able to suppress the constitutively high capacity for high affinity iron uptake associated with loss of the chromosomal copy of ScATM1, abrogate intra-mitochondrial iron hyperaccumulation, and restore mitochondrial respiratory function and cytochrome c levels. By comparison, AtATM1 only weakly suppresses the atm1 phenotype, and AtATM2 exerts little or no suppressive action but instead is toxic when expressed in this system. The differences between AtATM3 and AtATM1 are maintained after exchanging their target peptides, and these proteins as well as AtATM2 colocalize with the mitochondrial fluor MitoTracker Red when expressed in yeast as GFP fusions. Although its toxicity when heterologously expressed in yeast, except when fused with GFP, precludes the functional analysis of native AtATM2, a common function, mitochondrial export of Fe/S clusters or their precursors for the assembly of cytosolic Fe/S proteins, is inferred for AtATM3 and AtATM1.

Cardiolipin is associated with the terminal oxidase of an extremely halophilic archaeon

Membranes having an a high content of cardiolipin were isolated from an extremely halophilic archaeon Halorubrum sp. Absorbance difference spectra of detergent-solubilized plasma membranes reduced by dithionite suggested the presence of b-type cytochromes. Non-denaturing gel electrophoresis revealed only one fraction having TMPD-oxidase activity in which cardiolipin was the major lipid component. The electroeluted fraction showed a cytochrome c oxidase activity characterized by the reduced minus oxidized difference spectra as a terminal heme-copper oxidase. The cytochrome c oxidase activity of the archaeal cardiolipin-rich membranes was inhibited by the cardiolipin-specific fluorescent marker 10- N-nonyl acridine orange (NAO) in a dose-dependent manner. The results indicate that an archaeal analogue of cardiolipin is tightly associated to archaeal terminal oxidases and is required for its optimal functioning.

Direct Observation of Redox-Linked Histidine Protonation Changes in the Iron−Sulfur Protein of the Cytochromebc1Complex by ATR-FTIR Spectroscopy

The redox-linked protonation chemistry of the iron-sulfur protein (ISP) of the cytochrome bc(1) complex was studied by analysis of the pH dependencies of redox difference spectra using perfusion/electrochemically induced attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy. The ISP of Rhodobacter capsulatus within the intact cytochrome bc(1) complex was analyzed in a mutant form in which the midpoint potential of cytochrome c(1) was lower than that of the ISP. This was compared to a soluble domain of the ISP from the bovine bc(1) complex. Spectra of in situ bacterial and isolated bovine proteins differ markedly only in part of their amide I regions with the in situ protein having additional pH-dependent component(s). Apart from this, both in situ and isolated proteins exhibited the same pH-dependent IR features in reduced minus oxidized difference spectra. Specifically, at high pH, a strong H/D insensitive negative band appeared at 1447/1450 cm(-)(1), together with a peak at 1310 cm(-)(1), the change of a 1267/1255 cm(-)(1) peak/trough into a simple 1266 cm(-)(1) peak, and a trough at 1107 cm(-)(1). Comparison with spectra of model materials indicates that all four signals arise from an imidazolate to imidazole transition of histidine, hence providing the first direct demonstration that histidine is the redox-linked protonation site of the ISP. The 1450 cm(-)(1) band can be assigned to a ring stretch that is unique to the imidazolate form of histidine. It is relatively sharp, has a high extinction coefficient, and provides a novel marker band for the detection of imidazolate intermediates in enzymatic mechanisms generally.

ATR-FTIR Spectroscopy and Isotope Labeling of the PM Intermediate of Paracoccus denitrificans Cytochrome c Oxidase

The structure of the P(M) intermediate of Paracoccus denitrificans cytochrome c oxidase was investigated by perfusion-induced attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy. Transitions from the oxidized to P(M) state were initiated by perfusion with CO/oxygen buffer, and the extent of conversion was quantitated by simultaneously monitoring visible absorption changes. In prior work, tentative assignments of bands were proposed for heme a(3), a change in the environment of the protonated state of a carboxylic acid, and a covalently linked histidine-tyrosine ligand to Cu(B) that has been found in the catalytic site. In this work, reduced minus oxidized difference spectra at pH 6.5 and 9.0 and P(M) minus oxidized difference spectra at pH 9.0 were compared in unlabeled, universally (15)N-labeled, and tyrosine-ring-d(4)-labeled proteins to improve these assignments. In the reduced minus oxidized difference spectrum, (15)N labeling resulted in large changes in the amide II region and a 9 cm(-1) downshift in a 1105 cm(-1) trough that is attributed to histidine. In contrast, changes induced by tyrosine-ring-d(4) labeling were barely detectable where the isotope-sensitive bands are expected. Both isotope substitutions had large effects on P(M) minus oxidized difference spectra. A prominent trough at 1542 cm(-1) was shifted to 1527 cm(-1) with (15)N labeling, and its magnitude was diminished with the appearance of a 1438 cm(-1) trough with tyrosine-ring-d(4) labeling. Both isotope substitutions also had large effects on a 1314 cm(-1) trough in the same spectra. These shifts indicate that the bands are linked to both a nitrogenous compound and a tyrosine, the most obvious candidate being the covalent histidine-tyrosine ligand of Cu(B). Comparison with model material data suggests that the tyrosine hydroxyl group is protonated when the binuclear center is oxidized but deprotonated in the P(M) intermediate. Positive bands at 1519 and 1570 cm(-1) were replaced with bands at 1504 and 1556 cm(-1), respectively, with tyrosine-ring-d(4) labeling, are characteristic of upsilon(7a)(C-O) and upsilon(C-C) bands of neutral phenolic radicals, and most likely reflect the formation of the neutral radical state of the histidine-tyrosine ligand in P(M).

Characterization of the cydAB-encoded cytochrome bd oxidase from Mycobacterium smegmatis.

The cydAB genes from Mycobacterium smegmatis have been cloned and characterized. The cydA and cydB genes encode the two subunits of a cytochrome bd oxidase belonging to the widely distributed family of quinol oxidases found in prokaryotes. The cydD and cydC genes located immediately downstream of cydB encode a putative ATP-binding cassette-type transporter. At room temperature, reduced minus oxidized difference spectra of membranes purified from wild-type M. smegmatis displayed spectral features that are characteristic of the gamma-proteobacterial type cytochrome bd oxidase. Inactivation of cydA or cydB by insertion of a kanamycin resistance marker resulted in loss of d-heme absorbance at 631 nm. The d-heme could be restored by transformation of the M. smegmatis cyd mutants with a replicating plasmid carrying the highly homologous cydABDC gene cluster from Mycobacterium tuberculosis. Inactivation of cydA had no effect on the ability of M. smegmatis to exit from stationary phase at 37 or 42 degrees C. The growth rate of the cydA mutant was tested under oxystatic conditions. Although no discernible growth defect was observed under moderately aerobic conditions (9.2 to 37.5 x 10(2) Pa of pO(2) or 5 to 21% air saturation), the mutant displayed a significant growth disadvantage when cocultured with the wild type under extreme microaerophilia (0.8 to 1.7 x 10(2) Pa of pO(2) or 0.5 to 1% air saturation). These observations were in accordance with the two- to threefold increase in cydAB gene expression observed upon reduction of the pO(2) of the growth medium from 21 to 0.5% air saturation and with the concomitant increase in d-heme absorbance in spectra of membranes isolated from wild-type M. smegmatis cultured at 1% air saturation. Finally, the cydA mutant displayed a competitive growth disadvantage in the presence of the terminal oxidase inhibitor, cyanide, when cocultured with wild type at 21% air saturation in an oxystat. In conjunction with these findings, our results suggest that cytochrome bd is an important terminal oxidase in M. smegmatis.

Study of cytochrome bo function in Vitreoscilla using a cyo(-) knockout mutant.

The bacterium, Vitreoscilla, produces a delta mu(Na+) across its membrane during respiration. A key enzyme for this function is the cytochrome bo terminal oxidase which, when incorporated into synthetic proteoliposomes, pumps Na(+) across the membrane upon the addition of a substrate. A Vitreoscilla cytochrome bo knock out (cyo(-)) mutant was isolated by transposon mutagenesis using pUT-mini-Tn5Cm. The membranes of this mutant lacked the characteristic 416 nm peak and 432 nm trough in CO difference spectra, which are clearly visible in spectra of the Vitreoscilla wild-type, but peaks at 627, 560, and 530 nm in reduced minus oxidized difference spectra indicate that cytochrome bd is still present. The specific NADH oxidase and ubiquinol-1 oxidase activities of the cyo(-) mutant membranes were less than those of Vitreoscilla wild-type and Escherichia coli membranes, and the stimulation of these activities of the mutant and E. coli membranes by 75 mM NaCl was approximately 50% less than that of Vitreoscilla wild-type membranes. The ubiquinol-1 oxidase activity of the cyo(-) mutant membranes was inhibited by 10 mM KCN to a lesser degree than that of the Vitreoscilla wild-type and E. coli membranes (50, 80, and 85%, respectively). This result is also consistent with the cyo(-) mutant membrane fragments containing only the cytochrome bd terminal oxidase, which is known to be less sensitive to KCN. Although the maximum respiration and growth of the cyo(-) mutant were less than those of the wild-type, this mutant is still capable of growing with cytochrome bd alone.

A Cytochrome bb′-type Quinol Oxidase inBacillus subtilis Strain 168

The aerobic respiratory system of Bacillus subtilis 168 is known to contain three terminal oxidases: cytochrome caa(3), which is a cytochrome c oxidase, and cytochrome aa(3) and bd, which are quinol oxidases. The presence of a possible fourth oxidase in the bacterium was investigated using a constructed mutant, LUH27, that lacks the aa(3) and caa(3) terminal oxidases and is also deficient in succinate:menaquinone oxidoreductase. The cytochrome bd content of LUH27 can be varied by using different growth conditions. LUH27 membranes virtually devoid of cytochrome bd respired with NADH or exogenous quinol as actively as preparations containing 0.4 nmol of cytochrome bd/mg of protein but were more sensitive to cyanide and aurachin D. The reduced minus oxidized difference spectra of the bd-deficient membranes as well as absorption changes induced by CO and cyanide indicated the presence of a "cytochrome o"-like component; however, the membranes did not contain heme O. The results provide strong evidence for the presence of a terminal oxidase of the bb' type in B. subtilis. The enzyme does not pump protons and combines with CO much faster than typical heme-copper oxidases; in these respects, it resembles a cytochrome bd rather than members of the heme-copper oxidase superfamily. The genome sequence of B. subtilis 168 contains gene clusters for four respiratory oxidases. Two of these clusters, cta and qox, are deleted in LUH27. The remaining two, cydAB and ythAB, encode the identified cytochrome bd and a putative second cytochrome bd, respectively. Deletion of ythAB in strain LUH27 or the presence of the yth genes on plasmid did not affect the expression of the bb' oxidase. It is concluded that the novel bb'-type oxidase probably is cytochrome bd encoded by the cyd locus but with heme D being substituted by high spin heme B at the oxygen reactive site, i.e. cytochrome b(558)b(595)b'.

The reduced minus oxidized difference spectra of cytochromes a and a3

We have re-investigated the reduced minus oxidized difference spectra of the two heme centers of cytochrome oxidase, cytochromes a and a 3. In contrast to data obtained in an earlier study (Vanneste, W.H. (1966) Biochemistry 5, 838–848), we find that the spectrum for cytochrome a 3 agrees with that found with a 5-coordinate high-spin heme A model compound. Small but significant additional differences are noted for both heme centers.

Isolation and characterization of a novel facultatively alkaliphilic bacterium, Corynebacterium sp., grown on n-alkanes

A novel facultatively alkaliphilic bacterium that grows on a chemically defined medium containing n-alkanes as the sole carbon source was isolated from soil. The isolate was obligately aerobic, non-motile, gram-positive, and formed metachromatic granules. It was not acidfast and did not form endospores. The cell wall contained meso-diaminopimelic acid, arabinose, and galactose; the glycan moiety of the cell wall contained acetyl residues. The bacterium was catalase-positive, oxidasenegative, and the G+C content of DNA was 70.8 mol%. According to these tests, the isolate was assigned to the genus Corynebacterium. The bacterium grew well between pH 6.2 to 10.2 and the doubling time in this pH range was 4–6 h. For the growth of the isolate, added Na+ in the culture medium stimulated growth, but was not indispensable at both pH 7.2 and pH 10.2. In addition to hydrocarbons, the isolate was able to grow on a chemically defined medium containing acetate, glucose, or fructose as the sole carbon source. Analysis of reduced minus oxidized difference spectra of whole cells showed that the bacterium only possessed less than one tenth the amount of total cytochromes as compared with Bacillus alcalophilus. The above results sugest that the bacterium has characteristics different than those of the alkaliphilic Bacillus previously described.

Diphenyleneiodonium inhibits reduction of iron-sulfur clusters in the mitochondrial NADH-ubiquinone oxidoreductase (Complex I).

Diphenyleneiodonium (DPI) inhibits the mitochondrial NADH-ubiquinone oxidoreductase (Complex I) on the substrate side of the Fe-S clusters. In the inhibited NADH-supplemented state all of the Fe-S clusters are oxidized, whereas the reduced minus oxidized difference spectrum of the protein-bound FMN can be visualized. It is characterized by troughs at 370 and 450 nm and a small increase of absorbance in the 500-700-nm region. DPI probably reacts irreversibly with FMN, because oxidation of FMN is blocked even after its extraction from the enzyme. Inhibition requires preincubation of enzyme in the presence of NADH and DPI. The lower the NADH/NAD+ ratio or the pH, or the higher the NAD+/DPI ratio, the more DPI is required for inhibition. NAD+ and DPI apparently compete for a common site. Both ubiquinone and dichlorophenolindophenol reductase activities are fully blocked by DPI, whereas the ferricyanide reductase activity is inhibited by 75%. Similar results were found with Complex I and two rotenone-insensitive preparations, subcomplex I lambda and the flavoprotein fraction. DPI also inhibits NADH oxidation by bacterial NADH-ubiquinone oxidoreductase-1 (NDH-1) in membranes of Paracoccus denitrificans and Escherichia coli.

Identification of a new b-type cytochrome from the whitefish Coregonidae eggs

A new b-type cytochrome, termed cytochrome b 560 according to the wavelength maximum of its α-band in the reduced minus oxidized difference spectrum, was isolated from eggs of whitefish and partially purified by fractionation of the water-soluble moiety of yolk. The absolute absorption spectrum of reduced cytochrome b 650 has maxima at 426, 529 and 560 nm and that of the oxidized form at 416 nm. The reduced minus oxidized difference spectrum has maxima at 428, 529 and 560 nm. The midpoint potential of this cytochrome is +193 mV. Based on the MCD spectra of reduced cytochrome and the optical absorption spectra in the visible region of the oxidized cytochrome, it is suggested that the heme iron in cytochrome b 560 has two histidine imidazoles as the 5th and 6th axial ligands.

Cytochromes in Thiobacillus tepidarius and the respiratory chain involved in the oxidation of thiosulphate and tetrathionate

Thiobacillus tepidarius was shown to contain cytochrome(s) c with absorption maxima at 421, 522 and 552 nm in room temperature reduced minus oxidized difference spectra, present at 1.1–1.2 nmol per mg dry wt and present in both membrane and soluble fractions of the cell. The membrane-bound cytochrome c (1.75 nmol per mg membrane protein) had a midpoint potential (Em, pH 7.0) of 337 mV, while the soluble fractions appeared to contain cytochrome(s) c with Em (pH 7.0) values of about 270 and 360 mV. The organism also contained three distinct membrane-bound b-type cytochromes (totalling 0.33 nmol per mg membrane protein), each with absorption maxima in reduced minus oxidized difference spectra at about 428, 532 and 561 nm. The Em (pH 7.0) values for the three cytochromes b were 8 mV (47.8% of total), 182 mV (13.7%) and 322 mV (38.5%). No a- or d-type cytochromes were detectable spectrophotometrically in the intact organism or its membrane and soluble fractions. Evidence is presented for both CO-binding and CO-unreactive cytochromes b or o, and CO-binding cytochrome(s) c. From redox effects observed with CO it is proposed that a cytochrome c donates electrons to a cytochrome b, and that a high potential cytochrome b or o may be acting as the terminal oxidase in substrate oxidation. This may be the ‘445 nm pigment’, a photodissociable CO-binding membrane haemoprotein. Substrate oxidation was relatively insensitive to CO-inhibition, but strongly inhibited by cyanide and azide. Thiosulphate oxidation couples directly to cytochrome c reduction, but tetrathionate oxidation is linked (probably via ubiquinone Q-8) to reduction of a cytochrome b of lower potential than the cytochrome c. The nature of possible electron transport pathways in Thiobacillus tepidarius is discussed. One speculative sequence is: c− → b8 → b182 → c270 → c337 → b322/c360 → O2

Production of recombinant cytochrome b558 allows reconstitution of the phagocyte NADPH oxidase solely from recombinant proteins

Phagocytic white blood cells contain a multicomponent oxidase that generates microbicidal products by catalyzing electron transfer from NADPH to molecular oxygen. Activation of this oxidase requires interactions of a unique membrane flavocytochrome with the cytosolic proteins p47phox, p67phox, and p21Rac. This flavocytochrome, designated cytochrome b558, is a heteromer comprising a 22-kDa alpha-subunit (p22phox) and a glycosylated approximately 91-kDa beta-subunit (gp91phox). Cytochrome b558 was expressed in Sf9 insect cells coinfected with recombinant baculoviruses carrying cDNAs for p22phox and gp91phox. Membranes of these cells contained a b-type cytochrome with a dithionite-reduced minus oxidized difference spectrum similar to that of neutrophil cytochrome b558. The recombinant cytochrome b558 beta-subunit was heterogeneously N-glycosylated as demonstrated by its susceptibility to cleavage with endoglycosidases F and H. In contrast to the neutrophil cytochrome b558, a portion of the N-linked oligosaccharide was of the high mannose type. Recombinant cytochrome b558 supported superoxide production in a cell-free assay containing recombinant p47phox, p67phox, and p21Rac. The enzymatic turnover of the partially purified recombinant cytochrome b558 and neutrophil cytochrome b558 were similar (approximately 100-160 mol of superoxide generated/s/mol of cytochrome heme, range of two experiments) and the native and recombinant cytochromes showed similar requirements for NADPH and exogenous FAD. These studies represent the first reconstitution of the NADPH oxidase solely from recombinant proteins and define a model system to explore the structure and function of cytochrome b558.

Mitochondria of mammalianPlasmodiumspp.

Highly purified mitochondrial fractions have been isolated from the intraerythrocytic stages of two mammalian Plasmodium spp., Plasmodium yoelii of rodents and Plasmodium falciparum of man. Mitochondria of the former parasite are cristate whereas those of the latter are essentially acristate. Isolated mitochondria from both parasite species were heterogeneous with respect to size, shape, density of matrix staining and extent of internal structure. Respiratory assay, by reduction of exogenous cytochrome c, showed NADH, alpha-glycerophosphate and succinate to be the substrates with the greatest potential for metabolism. Additionally, proline, dihydroorotate and glutamate (P. falciparum only) were oxidized at low rates. A number of NAD(+)-linked substrates were not utilized. The NADH-dependent reduction of cytochrome c was insensitive to rotenone and antimycin A. Fumarate inhibited the NADH-dependent reduction of cytochrome c and stimulated the oxidation of NADH, suggestive of an NADH-fumarate reductase pathway. Oxidation of either alpha-glycerophosphate or succinate was fully inhibited by standard mitochondrial electron transport inhibitors, including a number of Complex III inhibitors, although the concentrations required of such inhibitors (notably myxothiazol) were relatively high compared to mammalian mitochondria. Dithionite-reduced minus oxidized difference spectra indicated the presence of cytochromes aa3, b, c and c1 in mitochondria of both parasite species, but at a higher cytochrome to protein ratio in P. yoelii. Freshly isolated mitochondria from either species exhibited only low respiratory control ratios with alpha-glycerophosphate or succinate as substrates. The apparent absence of a respiratory chain 'Site I' in such mitochondria may mean that NADH-fumarate reductase serves to reoxidize mitochondrial NADH.

The occurrence and function of a-type cytochromes in the aerobic respiratory chain of Comamonas percolans NCTC 1937 grown under O2-sufficient and O2-limited conditions

Intact cells of Comamonas percolans (NCTC 1937) after growth under O2-limited conditions in batch culture exhibit, in addition to b- and c-type cytochromes, an unusual pigment absorbing variably at 588 to 594 nm in reduced minus oxidized difference spectra at room temperature. CO difference spectra suggest ligand binding by this pigment and also reveal a prominent 448 nm absorption minimum and CO-binding b- and/or c-type cytochromes. Although the 588 to 594 nm-absorbing component is reminiscent of ‘cytochrome a 1’, claimed to be a terminal oxidase in some bacteria, O2-limited cells of C. percolans contain no detectable haem A. In contrast, cells grown under O2-sufficient conditions exhibit a membrane-bound cytochrome aa 3 and contain haem A. Low-temperature photodissociation studies of O2-sufficient cells show cytochrome aa 3 to be functional in CO and O2 binding and suggest that aa 3 is the terminal oxidase of a cytochrome b- and c-containing aerobic respiratory chain. Analogous studies of O2-limited cells reveal a component absorbing, in its unliganded state, at 448 nm. Exposure of such CO-liganded cells to white actinic light is followed by oxidation of b- and/or c-type cytochromes and, although the functional oxidase has not been identified, we conclude that C. percolans does not utilize a cytochrome oxidase of the a 1 type.

Inhibition of mitochondrial respiratory chain by arylthiolated 2,3-ethylenedioxy-1,4-benzoquinones.

A series of arylthiolated 2,3-ethylenedioxy-1,4-benzoquinones as a coenzyme Q (CoQ) antagonist was tested for inhibition of succinate oxidase and reduced nicotinamide adenine dinucleotide (NADH) oxidase systems in the mitochondrial respiratory chain. The following characteristics were revealed: (1) 2,3-ethylenedioxy, 5-arylthio and 5,6-diarylthio groups were confirmed to be favorable for inhibition of both systems; (2) these analogs were more effective in the succinate oxidase system than in the NADH oxidase system; (3) 4' substituents on the benzene side ring had little effect on inhibitory activity; (4) the acting sites of these analogs had no strict stereospecificity. The reduced minus oxidized difference spectra revealed that these analogs inhibited the succinate oxidase system at the site between succinate and CoQ, and the NADH oxidase system at the site after cytochrome a + a3, suggesting these analogs might act as antagonists of CoQ in the succinate oxidase system. However, 5-(4'-nitrophenylthio)-2,3-ethylenedioxy-1,4-benzoquinone (If) strongly inhibited only the succinate oxidase system at the site after cytochrome a + a3.