CO-binding Cytochrome Research Articles

Effect of Growth Conditions on the Synthesis of Terminal Oxidases in Methylobacillus flagellatus KT

Membranes of the obligate methylotroph Methylobacillus flagellatus KT contained hemes B, O, and C and cytochromes b, o, and c both in batch and in continuous cultures. Neither heme A nor heme D was detected in the membranes. The cytochromes o and bb were the main components reversibly binding carbon monoxide (CO) in the terminal part of the respiratory chain. The α-region and especially the α-peaks at 568 and 573 nm and the α-troughs at 586 and 592 on the CO-difference spectra were diagnostic for the cytochromes o and bb, respectively. The cytochrome o content increased up to 1.8 times upon increasing the dilution rate of the culture from 0.15 to 0.55 h−1 under methanol limitation. By contrast, the level of the CO-binding cytochrome bb was not affected by methanol concentration but its content increased up to 1.9 times when the level of oxygen decreased from 95 to 21 μM under the constant dilution rate (μ = 0.55 h−1). The maximum ratio between the cytochromes o and bb reached 2 during continuous cultivation under methanol-limited conditions (μ = 0.55 h−1), whereas the minimum ratio between them was about 0.7 during batch cultivation at stationary phase of growth. The synthesis of the CO-binding cytochrome bb but not of the cytochrome o in M. flagellatus KT was assumed to depend on the ambient redox potential of the medium. The cytochrome o synthesis was supposed to depend on the transmembrane gradient of protons (ΔμH+).

Lithoautotrophic growth of the freshwater strain Beggiatoa D-402 and energy conservation in a homogeneous culture under microoxic conditions.

The freshwater filamentous bacterium Beggiatoa D-402 was shown to grow lithoautotrophically in a homogeneous culture under microoxic conditions only, the growth yield being the highest at 0.1 mg O(2) l(-1). High activities of the Calvin cycle key enzymes and of the dissimilatory path thiosulfate oxidation enzymes were found in the bacterial cells. The rate of CO(2) fixation above 112 nmol min(-1) (mg protein)(-1), an about 90% increase in the protein carbon at the expense of CO(2) carbon and an increase in the molar yield up to 12 mg dry weight (mmol oxidized thiosulfate)(-1) indicate the bacterial growth was autotrophic. Thiosulfate was oxidized by the strain almost completely into sulfate. The metabolically useful energy was conserved by oxidative phosphorylation that was coupled to oxidation of sulfur compounds. The bacterial membranes were found to contain CO-binding cytochromes b and two cytochromes c with M(r) 23 and 26 kDa, the terminal part of the respiratory chain containing presumably a cbb(3)-type oxidase. A cytochrome c with M(r) 12 kDa was detected in the soluble fraction.

Lithoautotrophic growth of the freshwater strain Beggiatoa D-402 and energy conservation in a homogeneous culture under microoxic conditions

The freshwater filamentous bacterium Beggiatoa D-402 was shown to grow lithoautotrophically in a homogeneous culture under microoxic conditions only, the growth yield being the highest at 0.1 mg O2 l−1. High activities of the Calvin cycle key enzymes and of the dissimilatory path thiosulfate oxidation enzymes were found in the bacterial cells. The rate of CO2 fixation above 112 nmol min−1 (mg protein)−1, an about 90% increase in the protein carbon at the expense of CO2 carbon and an increase in the molar yield up to 12 mg dry weight (mmol oxidized thiosulfate)−1 indicate the bacterial growth was autotrophic. Thiosulfate was oxidized by the strain almost completely into sulfate. The metabolically useful energy was conserved by oxidative phosphorylation that was coupled to oxidation of sulfur compounds. The bacterial membranes were found to contain CO-binding cytochromes b and two cytochromes c with Mr 23 and 26 kDa, the terminal part of the respiratory chain containing presumably a cbb3-type oxidase. A cytochrome c with Mr 12 kDa was detected in the soluble fraction.

Characterization of the genes encoding the three-component membrane-bound alcohol dehydrogenase from Gluconobacter suboxydans and their expression in Acetobacter pasteurianus.

The three-component membrane-bound alcohol dehydrogenase (ADH) of Gluconobacter suboxydans IFO12528 was purified, and the NH2-terminal amino acid sequence of each subunit was determined. On the basis of the amino acid sequences, the genes adhA, encoding the 72-kDa dehydrogenase, adhB, encoding the 44-kDa cytochrome c-553 (a CO-binding cytochrome c), and adhS, encoding a 15-kDa protein, were cloned and the amino acid sequences of their products were deduced from the nucleotide sequences. The dehydrogenase and cytochrome genes were clustered with the same transcription polarity, as is the case in species of Acetobacter, another genus of acetic acid bacteria. These AdhA and AdhB subunits showed similarity in amino acid sequence to those from Acetobacter spp., whereas AdhS showed no similarity to the corresponding subunit of the ADH complex of Acetobacter pasteurianus. Consistent with this, adhS of G. suboxydans could not complement a defect in the corresponding subunit of A. pasteurianus. When the adhA-adhB gene cluster of G. suboxydans was expressed in an ADH-deficient mutant of A. pasteurianus, the transformant showed distinct ADH activity. The ADH complex was purified to near homogeneity and consisted of two subunits, the dehydrogenase and the cytochrome c subunits derived from G. suboxydans, without any other subunit. These data suggested that AdhS, the smallest subunit of ADH, from G. suboxydans is not essential for ADH activity in A. pasteurianus, in contrast to the essential role of A. pasteurianus AdhS, which is required for correct assembly of the dehydrogenase and cytochrome c subunits on the membrane.

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

Presence of functional cytochrome P-450 on isolated rat hepatocyte plasma membrane

Antibodies against cytochrome P-450 are found in some children with autoimmune hepatitis (antiliver/kidney microsome 1) and in patients with ticrynafen hepatitis (antiliver/kidney microsome 2). For an immune reaction against cytochrome P-450 to possibly destroy the hepatocytes, one must assume that cytochrome P-450 is present on the plasma membrane surface of hepatocytes. In a first series of experiments, plasma membranes were prepared with a technique based on the electrostatic attachment of isolated hepatocytes to polyethyleneimine-coated beads. After vortexing, beads were coated with a very pure plasma membrane fraction. Microsomal contamination, judged from the specific activities of glucose-6-phosphatase or NADH-cytochrome c reductase, was less than 1%. Nevertheless, the specific content (per milligram of protein) of CO-binding cytochrome P-450 was 20% of that in microsomes; the specific benzo(a)pyrene hydroxylase activity was 25%, and ethoxycoumarin deethylase 11%. Immunoblots showed the presence of cytochromes P-450 UT-A, UT-H, PB-B, ISF-G and PCN-E, the last three isoenzymes being inducible by, respectively, phenobarbital, 3-methylcholanthrene and dexamethasone. In a second series of experiments, nonpermeabilized isolated hepatocytes from untreated rats were incubated with anticytochrome P-450 antibodies. Immunofluorescence and immunoperoxidase staining confirmed the presence of cytochromes P-450 UT-A, PB-B and ISF-G on the membrane. In a last series of experiments, human antiliver-kidney microsomal 1 antibodies were found to react specifically with rat liver plasma membrane cytochrome P-450 UT-H (IID subfamily). We conclude that several cytochrome P-450 isoenzymes are present, active and inducible on the plasma membrane surface of hepatocytes. It is therefore conceivable that immunization against plasma membrane cytochrome P-450 might lead to the immunological destruction of hepatocytes in some patients.

Presence of Functional Cytochrome P–450 on Isolated Rat Hepatocyte Plasma Membrane

Antibodies against cytochrome P-450 are found in some children with autoimmune hepatitis (antiliver/kidney microsome 1) and in patients with ticrynafen hepatitis (antiliver/kidney microsome 2). For an immune reaction against cytochrome P-450 to possibly destroy the hepatocytes, one must assume that cytochrome P-450 is present on the plasma membrane surface of hepatocytes. In a first series of experiments, plasma membranes were prepared with a technique based on the electrostatic attachment of isolated hepatocytes to polyethyleneimine-coated beads. After vortexing, beads were coated with a very pure plasma membrane fraction. Microsomal contamination, judged from the specific activities of glucose-6-phosphatase or NADH-cytochrome c reductase, was less than 1%. Nevertheless, the specific content (per milligram of protein) of CO-binding cytochrome P-450 was 20% of that in microsomes; the specific benzo(a)pyrene hydroxylase activity was 25%, and ethoxycoumarin deethylase 11%. Immunoblots showed the presence of cytochromes P-450 UT-A, UT-H, PB-B, ISF-G and PCN-E, the last three isoenzymes being inducible by, respectively, phenobarbital, 3-methylcholanthrene and dexamethasone. In a second series of experiments, nonpermeabilized isolated hepatocytes from untreated rats were incubated with anticytochrome P-450 antibodies. Immunofluorescence and immunoperoxidase staining confirmed the presence of cytochromes P-450 UT-A, PB-B and ISF-G on the membrane. In a last series of experiments, human antiliver-kidney microsomal 1 antibodies were found to react specifically with rat liver plasma membrane cytochrome P-450 UT-H (IID subfamily). We conclude that several cytochrome P-450 isoenzymes are present, active and inducible on the plasma membrane surface of hepatocytes. It is therefore conceivable that immunization against plasma membrane cytochrome P-450 might lead to the immunological destruction of hepatocytes in some patients.

Effects of low environmental manganese concentrations on the respiratory chains of Paracoccus denitrificans

Paracoccus denitrificans was cultivated in continuous culture in media deficient in manganese, or copper or both metals and the effects on growth and the respiratory chains studied. Manganese deficiency had no effect on growth yield or the levels of cytochrome c oxidase but the amount of c-type cytochromes was decreased to about 35% of control values and a CO-binding b-type cytochrome was also produced. However, the major functioning oxidase was still cytochrome c oxidase. The ESR and optical signals of copper were present or absent if cells were grown in the presence or absence of copper respectively, independent of the concentration of manganese available. This allows the ESR properties of the native and copper-deficient cytochrome c oxidase to be studied as the spectra are now not obscured by signals from manganese.

Mutations Affecting the Cytochrome d-Containing Oxidase Complex of Escherichia coli K12: Identification and Mapping of a Fourth Locus, cydD

A mutant of Escherichia coli K12 has been isolated affected in a gene, designated cydD, distinct from the three previously described loci involved in the synthesis of assembly of the cytochrome bd oxidase complex. The mutant, obtained by nitrosoguanidine mutagenesis, lacks the spectroscopically detectable components of this oxidase, namely cytochromes b558, b595 and d. Cytochrome oxidase o is the sole CO-binding cytochrome in membranes of the mutant, but the soluble haemoprotein b-590 and catalase activity appear unaffected. Discrimination between Cyd+ and Cyd- strains is facilitated by the development of a defined low-phosphate medium that allows the inclusion of Zn2+ as well as azide, inhibitors of respiratory electron transfer particularly via cytochrome o. Mapping with F-prime factors and by P1 cotransductional frequencies shows the mutation to map near 19.3 min on the E. coli chromosome, distinct from cydC, which maps at 18.9 min. The gene order in this region was tested in a three-factor cross and demonstrates the order zbj::Tn10(YYC199)-cydD-aroA, consistent with cotransduction frequencies.

Effects of copper concentration in continuous culture on the aa 3-type cytochrome oxidase and respiratory chains of Paracoccus denitrificans

The role(s) of copper in a bacterial cytochrome oxidase of the aa3-type was investigated by growth of Paracoccus denitrificans NCIB 8944, in batch and steady state continuous culture, in a medium from which the bulk of the copper had been extracted. In a medium containing approximately 0.02 μM copper, cellular copper content, cytochromes a+a3 and cytochrome a3 were reduced to 55%, 58% and 33% respectively of control values and there were also less marked decreases in cytochromes c+c1 (to 85%) and a CO-binding b-type cytochrome, possibly cytochrome o (to 71%). Copper deficiency elicited in reduced minus oxidized difference spectra a shift to shorter wavelengths and narrowing of the band width of the α-band of the oxidase, and loss of a (negative) band near 830 nm attributable to CuA (the copper functionally associated with haem a in the oxidase complex). The oxidase in copper-deficient cells reacted with oxygen to form the oxy “Compound A” at rates similar to that in control cells but CO recombination to ferrous haem a3 was slowed 4-fold in the copper deficient case. The results are interpreted as indicating loss of CuA and changes in the proportions of haems a and a3 with retention of catalytic activity. Titrations of respiration rates with antimycin suggested that copper deficiency did not result in diversion of electron flux through an antimycin A-insensitive, cytochrome o-terminated branch of the respiratory chain.

Inhibitory effects of nilutamide, a new androgen receptor antagonist, on mouse and human liver cytochrome P-450

The effects of nilutamide were studied first with human liver microsomes. At concentrations expected in the human liver (110 μM), nilutamide inhibited hexobarbital hydroxylase, benzphetamine N-demethylase, benzo( a)pyrene hydroxylase and 7-ethoxycoumarin O-deethylase activities by 85, 40, 35 and 25%, respectively. There was no in vitro inhibition of NADPH-cytochrome c reductase activity, no in vitro loss of CO-binding cytochrome P-450, and no spectral evidence for the in vitro formation of a possible cytochrome P-450Fe(II)-nitroso metabolite complex. Other studies were performed with mouse liver microsomes. Nilutamide (550 μM) did not significantly increase the consumption of NADPH by aerobic microsomes, and did not modify the kinetics for the reduction of cytochrome P-450 by NADPH-cytochrome P-450 reductase in an anaerobic system. Nilutamide (22 μM) produced either a type I or a type II binding spectrum. Kinetics for the inhibition of hexobarbital hydroxylase were consistent with competitive inhibition. A last series of experiments was performed after administration of nilutamide in mice. Thirty minutes after administration of doses (15 or 30 ,umol. kg~ ' i.p.) similar to those used in humans, the hexobarbital sleeping time was increased by 40 and 60%, respectively. There was no evidence, however, for the irreversible inactivation of microsomal enzymes since CO-binding cytochrome P-450 and monooxygenase activities remained unchanged in liver microsomes from mice killed 1 or 6 hr after administration of nilutamide (30 μmol. kg −1.p.). These results show that nilutamide inhibits hepatic cytochrome P-450 activity, and suggest that inhibition may actually occur after therapeutic doses of nilutamide in humans.

Spectral and potentiometric analysis of cytochromes from Bacillus subtilis.

Bacillus subtilis cytoplasmic membranes contain several cytochromes which are linked to the respiratory chain. At least six different cytochromes have been separated and identified by ammonium sulphate fractionation and ion-exchange chromatography. They include two terminal oxidases with CO-binding properties and cyanide sensitivity. One of these is an aa3-type cytochrome c oxidase which has characteristic absorption maxima in the reduced-oxidized difference spectrum at 601 nm in the alpha-band and at 443 nm in the Soret band regions. In the alpha-band two separate electron transitions with Em = +205 mV and Em = +335 mV can be discriminated by redox potentiometric titration. The other CO-binding cytochrome c oxidase contains two cytochrome b components with alpha-band maxima at 556 nm and 559 nm. Cytochrome b556 can be reduced by ascorbate and has an Em + +215 mV, whereas cytochrome b559 has an Em = +140 mV. Furthermore a complex consisting of a cytochrome b564 (Em = +140 mV) associated with a cytochrome c554 (Em = +250 mV) was found. This cytochrome c554, which can be reduced by ascorbate, appears to have an asymmetrical alpha-peak and stains for heme-catalyzed peroxidase activity on SDS-containing polyacrylamide gels. A protein with a molecular mass of about 30 kDa is responsible for this activity. A cytochrome b559 (Em = +65 mV) appears to be an essential part of succinate dehydrogenase. Finally a cytochrome c550 component with an apparent mid-point potential of Em = +195 mV has been detected.

Inactivation of human liver cytochrome P-450 by the drug methoxsalen and other psoralen derivatives

The effects of psoralen derivatives on cytochrome P-450 have been studied in human liver microsomes. CO-binding cytochrome P-450 was decreased by 33% after 10 min of incubation with 1.5 mM EDTA, an NADPH-regenerating system and 20 μM methoxsalen (8-methoxypsoralen). No destruction of cytochrome P-450 was observed when either NADPH or methoxsalen was omitted. A similar (27%) decrease in CO-bincling required a 100-times higher concentration of allylisopropylacetamide (2 mM). The activities of 7-ethoxycoumarin deethylase and benzo( a)pyrene hydroxylase were decreased by about 50% in the presence of 12.5 μM methoxsalen. At this low concentration, neither cimetidine nor SKF 525-A or piperonyl butoxide had any significant inhibitory effect. Monooxygenase activities were also decreased in the presence of 12.5 μM bergapten (5-methoxypsoralen) or 12.5 μM psoralen, but not with 12.5 μM trioxsalen (trimethylpsoralen). CO-bincling cytochrome P-450 was not decreased after 10 min of incubation with 1.5 mM EDTA, an NADPH-regenerating system and 20 μM trioxsalen. We conclude that methoxsalen is an extremely potent suicide inhibitor of cytochrome P-450 in human liver microsomes. Bergapten and psoralen are also inhibitory whereas trioxsalen has little effects. In the latter derivative, a methyl group is attached on the furan ring and may hinder its metabolic activation and the inactivation of cytochrome P-450.

Cytochrome content in Azospirillum brasilense Sp 7 grown under aerobic and denitrifying conditions

Azospirillum brasilense Sp 7 was grown in batch cultures with O2, [Formula: see text], [Formula: see text], or N2O as final electron acceptor. There were marked differences in cytochrome composition depending on the O2 status of the culture and the electron acceptor utilized. Highly aerated cultures showed no [Formula: see text] reduction and the cytochrome composition (a soluble c-type and particulate aa3, b, and c cytochromes) was not affected by the presence of[Formula: see text]. Under low aeration, in the presence of [Formula: see text], nitrate reductase activity occurred and there was a significant increase in the soluble and particulate cytochromes. Particulate cytochrome b-556 was observed only under high aeration and the cytochrome aa3, observed under both high and low aeration, decreased as the O2 concentration decreased. A particulate CO-binding cytochrome of type o was observed in the cells grown under high aeration. Cytochrome cd nitrite reductase was observed only in the soluble fraction of [Formula: see text]-grown cultures, which also contained the highest concentrations of the soluble cytochrome c-548 and the particulate c-551. N2O-grown cultures showed b-560 and c-551 cytochromes in the particulate and the c-548 cytochrome in the soluble fraction.

A screening method for cytochrome P-450 organic peroxidase activity and application to hydrocarbon-degrading bacterial populations

A method to detect the expression of hemoproteins with organic hydroperoxide reducing activity was developed to screen bacterial populations isolated from heavy oils and oil sands. The method is based on the activity of cytochrome P-450 as catalyst in the reduction of cumene hydroperoxide by artificial electron donors. There was no cross-reactivity with true peroxidases involved in the reduction of hydrogen peroxide. Cross-reactivity with catalase could be eliminated with appropriate inhibitors but did not normally interfere with the detection method. A preliminary screen resulted in the isolation of Acinetobacter calcoaceticus and a range of Gram-positive bacteria with organic peroxidase activity. Carbon monoxide difference spectra of cell-free extracts of the isolates revealed the presence of a hydrocarbon-inducible cytochrome P-450 in Acinetobacter calcoaceticus and in coryneform and actinomycete bacteria. A CO-binding cytochrome of unknown type with a Soret absorption maximum at 424 nm and cumene hydroperoxidase activity was also detected in some strains.

Electron transport and respiration in Beggiatoa and Vitreoscilla

Seven strains of bacteria belonging to the Beggiatoa-Vitreoscilla group were studied for their respiratory activity and for the presence of electron transport conponents. All strains tested oxidized [1-14C] and [2-14C] acetate to 14CO2 at relatively high rates. All strains tested were N,N,N′,N′-tetramethylphenylenediamine (TMPD)-oxidase positive and contained spectra representing a-type and carbon monoxide-binding cytochromes. Most of the strains also contained spectra representing c-type and b-type cytochromes. Beggiatoa alba B18LD contained b-type, a-type, c-type and CO-binding cytochromes, the latter two being located in the 144,000 x g soluble fraction. B. alba also contained ubiquinone-8 as its only detectable quinone.

Fumarate reductase and other mitochondrial activities in Trypanosoma cruzi

Subcellular fractions obtained from Trypanosoma cruzi epimastigotes broken by freezing and thawing were assayed for fumarate reductase activity with reduced methyl viologen as electron donor and fumarate as electron acceptor under anaerobic conditions. Two distinct activities were detected: one in the mitochondrial membranes, 115 mU(mg protein) −1, accounting for 96% of the total and the other in the cytosol, 3 mU(mg protein) −1, accounting for 3% of the total. The activity of membrane-bound fumarate reductase correlated statistically with either the activity or the amount of mitochondrial markers such as succinate and NADH dehydrogenases, cytochromes b + c 558, cytochrome a 611 and 5,7-diene sterols in the obtained subcellular fractions (580 × g, 12 000 × g, and 105 000 × g sediments and supernatant). Mitochondrial fumarate reductase was inhibited by succinate, malonate, cyanide, and 2-thenoyltrifluoroacetone (TTFA); whereas the soluble enzyme was inhibited by succinate and not by TTFA. The 12 000 × g sediment (mitochondrial membranes) showed after dithionite addition, absorption maxima at 611, 560 and 530 nm accounting for the presence of cytochromes b 560, c 558 and a 611. A CO-binding cytochrome o was also detected. A scheme of the T. cruzi mitochondrial respiratory chain is presented.

Cytochromes in Chloroflexus aurantiacus grown with and without oxygen

The major types of cytochromes present in Chloroflexus aurantiacus strain J-10-f1 were determined by analysis of cell-free preparations and membranes from cells grown phototrophically and chemotrophically. Reduced minus oxidized difference spectroscopy as well as quantitative analysis of the pyridine ferrohemochromogens in alkaline solution were used to determine the classes of cytochromes present. Evidence for membrane-bound cytochromes of the b and c-type was found in cell-free preparations of phototrophically grown cells. Difference spectra revealed absorption maxima at 553 nm, 523 nm, and 418 nm and a shoulder at 562 nm. Protoheme and heme c were identified as alkaline pyridine ferrohemochromogens with absorption maxima at 556 and 550 nm respectively. The ratio of heme c to protoheme in the cell-free preparation was about 10 to 1 with 0.65 μmol of heme c and 0.067 μmol of protoheme per gram of total cell protein. Dithionite plus carbon monoxide minus dithionite reduced difference spectra of membranes revealed a peak at 414 nm and troughs at 552, 523, and 423 nm consistent with a CO-binding cytochrome c. Similar analyses of chemotrophically grown cells revealed evidence for the presence of membrane-bound cytochromes of the c, b, and a classes with absorption maxima at 552–557 and 593–596 nm. Protoheme, heme c and heme a were detected as pyridine ferrohemochromogens with maxima at 556, 550, and 588 nm respectively. The ratio of heme c to protoheme was about 3 to 1 with 0.44 μmol of heme c and 0.16 μmol of protoheme per gram of total cell protein.

Comparative studies on redox proteins from ammonia-oxidizing bacteria

Soluble fractions prepared from representatives of 3 genera of ammonia-oxidising bacteria all contained CO-binding c-type cytochromes and the cytochrome P-460 chromophore. Hydroxylamine oxidase from terrestrial strains of Nitrosomonas europaea was immunologically related to the analogous protein from marine strains of the same genus. Soluble cytochrome oxidase nitrite reductase activity was detected only in extracts prepared from terrestrial strains of N. europaea among the organisms examined.

The cytochrome composition of carboxydotrophic bacteria

Spectroscopy at room and liquid nitrogen temperatures with extracts of the carbon monoxide-oxidizing bacteria Pseudomonas carboxydovorans, P. carboxydohydrogena, P. carboxydoflava, P. compransoris, Alcaligenes carboxydus, and Arthrobacter 11/x revealed the presence of normal electron transport systems, containing b-, c-, and a-type cytochromes at concentrations that compare to those of other aerobic bacteria. CO did not induce the formation of special CO-insensitive terminal oxidases. The gross composition of the respiratory chains was not affected by the type of growth substrate, and cytochrome d(=a2) was not detected. However, certain b-type cytochromes were only found when CO or H2 + CO2 served as growth substrates. All strains contained at least two different b-type cytochromes. Cytochrome b563 formed a weak CO-complex and was identified as a novel cytochrome o. It functions as CO-insensitive, alternative terminal oxidase in carboxydotrophic bacteria. A soluble CO-binding cytochrome c was present in P. carboxydovorans, P. carboxydohydrogena, and P. carboxydoflava. A CO-binding protoheme compound could be identified as catalase in P. compransoris, P. carboxydovorans, P. carboxydohydrogena, A. carboxydus, and Arthrobacter 11/x. The data are consistent with the presence of branched respiratory chains in the carboxydotrophs examined, and suggest the functioning of both, cytochrome a and the novel cytochrome o as terminal oxidases.