Cytochrome Spectra Research Articles

Spectrophotometric analysis of yeasts. Cytochrome spectra of some Candida and related taxa.

Whole-cell pastes of eight Candida species and their suggested teleomorph counterparts in the genera Pichia and Issatchenkia analyzed were spectrophotometrically. All of these yeasts, included in the two very closely related groups krusei and norvegensis, showed strong similarities in their low-temperature cytochrome absorption spectra, especially in two typical peaks, generally at 597-597.5nm and 605-606.5nm or in some cases at 593.5-594.5nm and 601.5-602nm, in the spectral region of the α maximum absorption of cytochrome aa3. In addition, the two species of each assumed pair of perfect and imperfect yeasts, I. orientalis/C. krusei, I.occidentalis/C. sorbosa, P. fermentans/C. lambica, P. nakasei/C. citrea or P. norvegensis/C. norvegensis were indistinguishable. Cytochrome spectra also indicated that I. terricola may no longer be considered a teleomorph of C. diversa.

Effects of oxygen on the growth and metabolism ofActinomyces viscosus

Actinomyces viscosus is a predominant microorganism in dental plaque. It is, just as the oral Streptococcus spp., a saccharolytic and aero-tolerant organism. We have investigated the effects of oxygen on the growth and metabolism of A. viscosus . To this end A. viscosus Ut 2 was grown in a glucose limited chemostat culture on a chemically defined medium ( D = 0.2 h−1) with exposure to variable amounts of oxygen. The Yglucose increased from 62.5 g · mol−1 under anaerobic conditions to 149 g · mol−1 under aerobic conditions, while, concomitantly, the carbon recovery from acidic fermentation products decreased from 75% to 7%. Addition of [14C]glucose to the chemostat showed that the glucose, which was not converted to acidic fermentation products, was instead converted to carbon dioxide or used for the production of biomass. Under aerobic and anaerobic conditions identical cytochrome spectra, containing only two cytochrome b -type absorption bands, were found. It was concluded that electron transport phosphorylation probably occurs both under aerobic and anaerobic conditions. Anaerobically, fumarate served as the electron acceptor, while the high growth yields observed under aerobic conditions are likely to be explained by citric acid cycle activity coupled to electron transport phosphorylation.

The purification of cytochrome f and plastocyanin using affinity chromatography

Both plastocyanin and cytochrome f were purified using a combination of affinity chromatography together with established methods. Plastocyanin was partially purified using the method of Davis and San Pietro (Anal. Biochem. 95 (1979) 254–259), after which it was further purified using a column of cytochrome c covalently attached to Sepharose 4B. The affinity column was prepared using the method of Godinot and Gautheron (Methods Enzymol. 54 (1979) 112–114). The final purity index ratio ( A 278 A 597 ) was less than 1.2, which is equal to that obtained using the more expensive FPLC procedure (Anderson, G.P., Sanderson, D.G., Lee, C.H., Durell, S., Anderson, L.B. and Gross, E.L. (1987) Biochim. Biophys. Acta 894, issue 3). Cytochrome f was partially purified using a modification of the method of Matazaki et al. (Plant Cell. Physiol. 16 (1975) 237–246) and bound to an affinity column of plastocyanin covalently attached to Sepharose 4B. Cytochrome f purified using this procedure had a purity index ratio ( A 554.5 A 277 ) of 1.2. Both proteins are tyrosine proteins containing no tryptophan residues. After the affinity chromatography step, the fluorescence emission spectrum of either plastocyanin or cytochrome f was typical of a tyrosine protein free from tryptophan contamination.

Fluorescence line-narrowing spectra of Zn-cytochromec. Temperature dependence

Fluorescence site selection spectra of Zn-substituted cytochrome c and Zn-mesoporphyrin were measured as a function of excitation energy and temperature within 5–60 K. The emission energies determined for Zn-cytochrome c were comparable with those of Zn-mesoporphyrin and mesoporphyrin in polar glassy matrices and with resonance Raman spectra of heme proteins. In the site selection spectra of both Zn-cytochrome c and Zn-mesoporphyrin in polar organic glass an increase in the intensity of zero phonon emission lines was observed with an increase of temperature between 5 and 30 K. The irregular spectral changes were interpreted on the basis of the temperature dependence of the linewidth as well as possible hole-burning phenomena.

Spectroscopic analysis of the cytochrome c oxidase-cytochrome c complex: circular dichroism and magnetic circular dichroism measurements reveal change of cytochrome c heme geometry imposed by complex formation.

Binding of cytochrome c to cytochrome c oxidase induces a conformational change in both proteins as well as a change of the electronic structure of the heme of cytochrome c, indicating an altered heme c-protein interaction. This follows from the observation that the induced circular dichroism (CD) and magnetic circular dichroism (MCD) spectra of the oxidase-cytochrome c complex in the Soret region differ from the summed spectra of oxidase plus cytochrome c. Spectral changes occur in the complex composed of either the two ferric or the two ferrous hemoproteins. The difference CD and MCD signals saturate at a ratio of 1 heme c per heme aa3. The difference spectra are specific to the cognate complex. The results are interpreted to reflect a direct relationship between the recognition/binding step and the electron-transfer reaction. The conformational rearrangement induced in cytochrome c by cytochrome c oxidase consists of a structural rearrangement of the heme environment and possibly a change of the geometry of the heme iron-methionine-80 sulfur axial bond. This rearrangement may decrease the reorganizational free energy of electron transfer by adjusting the heme c geometry to a state between that of ferri- and ferrocytochrome c.

Identification of the ligand-exchange process in the alkaline transition of horse heart cytochrome c.

Magnetic-circular-dichroism (m.c.d.) spectra over the wavelength range 300-2000 nm at room temperature and at 4.2K of horse heart cytochrome c are reported at a series of pH values between 7.8 and 11.0, encompassing the alkaline transition. The effect of glassing agents on the e.p.r. spectrum at various pH values is also reported. Comparison of these results with spectra obtained for the n-butylamine adduct of soybean leghaemoglobin support the hypothesis that lysine is the sixth ligand in the alkaline form of horse heart cytochrome c. The m.c.d. and e.p.r. spectra of horse heart cytochrome c in the presence of 1-methylimidazole have also been examined. These studies strongly suggest that histidine-18, the proximal ligand of the haem, is the ionizing group that triggers the alkaline transition. Low-temperature m.c.d. and e.p.r. spectra are also reported for Pseudomonas aeruginosa cytochrome c551. It is shown that no ligand exchange takes place at the haem in this species over the pH range 6.0-11.3.

Spectral properties of cytochrome c′ from Rhodopseudomonas capsulata B100 and its CO complex

The spectral properties of cytochrome c' from photosynthetic bacterium Rhodopseudomonas capsulata (= Rhodobacter capsulatus) B100 and its CO complex are reported. The electronic absorption, MCD, and EPR spectra have been compared with those of the other cytochromes c' and horse heart cytochrome c. EPR and electronic spectral results for the ferric cytochrome c' suggest that the ground state of heme-iron(III) at neutral pH consists of a quantum mechanical admixture of an intermediate-spin and a high-spin state and that at pH 11.0 is in a high-spin state. In the MCD spectrum of the CO-ferrous cytochrome c', the MCD intensity in the Soret band region was much higher than that of CO complexes of hemoproteins with a protoheme. The differences in a stereochemistry of the sixth-coordination position is discussed.

Effect of oxyanions of sulfur onThiobacillus ferrooxidans: Ferrous ion oxidation, oxygen uptake, and cytochrome reduction

Ferrous ion oxidation byThiobacillus ferrooxidans was completely inhibited by 10 mM each of thiosulfate, sulfite, metabisulfite, bisulfite, and tetrathionate. The inhibition was enhanced in a low pH medium (pH 1.5 versus pH 2.5). Oxygen uptake measurements with Fe2+ as the electron donor confirmed the toxicity of thiosulfate, but also indicated its dependency on the concentration of Fe2+. Cytochrome spectra of intact cells ofT. ferrooxidans showed that metabisulfite, and thiosulfate to a lesser extent, directly reduced electron transport components, in contrast to no direct reduction of cytochromes by tetrathionate and sulfite.

The biochemistry and immunology of yeasts that produce true mycelia: Ambrosiozyma, Hormoascus, and Hyphopichia.

Ambrosiozyma, yeasts that produce true mycelia with transverse septa that communicate by a dolipore type septum, are compared with Hyphopichia and Hormoascus. This study includes intracellular oxidases, nitrite and nitrate reductases, guanine plus cytosine content in DNA (GC-content), cytochrome spectra (a+a3, b, c), coenzyme Q, and serological comparison by immunodiffusion and immunoelectrophoresis, with A. monospora and Hormoascus platypodis as references. The results support the division of the genus Ambrosiozyma into 2 groups based upon GC content, vitamin requirements, and serological homologies. Affinities between Hormoascus platypodis and A. philentoma (vitamin requirements, intracellular nitrite and nitrate reductases, β-galactosidase, serological homology, cytochrome spectra, and GC content) leads to the placing of Hormoascus platypodis in synonymy with Ambrosiozyma philentoma. Ambrosiozyma ambrosiae is compared with the other Ambrosiozyma and with the non-fermentative Pichia, and Hansenula silvicola. These comparisons show that A. ambrosiae has more affinity with the other Ambrosiozyma than with Pichia or Hansenula. Hyphopichia burtonii differs from the other species in regard to vitamin requirements, coenzyme Q8, α2 band of cytochrome c and serological homologies.

Induction of petite mutation with acriflavine and elevated temperature in Candida albicans.

A method of inducing respiratory deficient (petite) mutation in Candida albicans (which has been previously classed as a petite-negative yeast) and characteristics of some isolated mutants are reported. When grown at 42 degrees C in the presence of a cytoplasmic mutagen (acriflavine), C. albicans exhibited diauxie-like biphasic growth. Mutants which were unable to grow on a non-fermentable substrate, glycerol, were isolated from the above culture at a frequency of less than 1%. The simultaneous action of both acriflavine and a supraoptimal temperature of 42 degrees C was required to induce respiratory mutation. The respiratory mutants were separated into two groups: i. mutants exhibiting a complete cytochrome spectrum but with low respiratory activity and ii. those deficient in cytochrome aa3. Further characterization of their respiratory activity, colony morphology, mitochondrial morphology and growth manner supported the evidence that members of the latter group can be regarded as petite mutants.

Cytochrome c1 and b-type cytochrome with two heme centers in the photosynthetic membranes from Rhodopseudomonas palustris

The photosynthetic membranes from Rhodopseudomonas palustris contained one species of membrane-bound c-type cytochrome, presumably cytochrome c 1, and a b-type cytochrome with two heme centers. The molecular weight and midpoint potential of cytochrome c 1 were 30000 and 275 mV, respectively. The α peak of the reduced-minus-oxidized difference spectrum of cytochrome c 1 was at 552 nm. Molecular weight of the b-type cytochrome was 32000 and the cytochrome had two midpoint potentials of 60 mV and −55 mV. The α peaks of the reduced-minus-oxidized difference spectra of the high and low midpoint potential heme centers were at 560 and 562 nm, respectively. These results suggested that there was a cytochrome b- c 1 complex in Rps. palustris.

1H NMR studies of bacterial class I cytochromes c

Nuclear magnetic resonance (NMR) spectral properties of Rhodopseudomonas gelatinosa cytochrome c 550, Rhodospirillum tenue cytochrome c 553, and Rps. capsulata (strains S.L. and 2.3.1) cytochrome c 2 are reported. Spectra of cytochromes C 550 and C 553 are compared with those of Pseudomonad cytochrome c 551. The sequence homology between them was found to be reflected by similar NMR spectral properties for resonances of some invariant groups. Packing around a heme propionate was found to be similar, despite substitutions of amino acids interacting with the propionate, although a conformational difference in this region between cytochrome c 550 and c 553 appears likely. The two cytochromes c 2 have different sequences and redox potentials [Pettigrew et al., Biochim. Biophys. Acta 503, 509–523 (1979)], but the two amino acid substitutions only cause minor conformational changes that are probably rearrangements of side chains close to the substitution sites.

Molecular cloning of the yeast nuclear genes CBS1 and CBS2.

The yeast nuclear genes CBS1 and CBS2 are both required for translation of the mitochondrial COB transcripts. Here we report on the identification of two unique chromosomal DNA-sequences of 2 kb and 2.3 kb from yeast wild type gene banks which functionally complement cbs1 and cbs2 mutants, respectively. Disruption of the homologous DNA-fragments by insertion of the URA3 gene generates respiratory deficient cells which fail to complement the original mutants. Cells with these gene disruptions are phenotypically identical to the original cbs1 and cbs2 mutants with respect to cytochrome spectra and mitochondrial translation products. The results exclude the possibility that suppressor genes have been cloned and confirm the conclusion that both genes, CBS1 and CBS2, specifically are involved in translation of mitochondrial COB RNA.

The respiratory chain of the facultative thermophile, Bacillus coagulans

Two oxidases were found to be present in membranes from the facultative thermophile Bacillus coagulans grown at 55°C, compared to one in cells grown at 37°C. Cytochrome spectra and inhibitors of the respiratory chain identified them as cytochrome oxidases aa 3 and d. Both were present in membranes from 55°C grown cells, but only cytochrome oxidase aa 3 was found in membranes from 37°C grown cells. The presence of cytochrome d in 55°C grown cultures was found to be due to decreased oxygen tension and not to the high growth temperature. This was confirmed by (a) induction of cytochrome d at 37°C under conditions of oxygen limitation and (b) its repression at 55°C under conditions of high aeration and its subsequent induction on lowering the dissolved oxygen concentration in chemostat cultures. Two cytochromes b (λmax 558 and λmax 562) were present in both 37°C and 55°C grown cells. Results from the inhibition of substrate oxidation by membranes suggested different pathways of electron transport by the respiratory chain.

Resonance Raman study on cytochrome c peroxidase and its intermediate. Presence of the Fe(IV) = O bond in compound ES and heme-linked ionization.

Resonance Raman spectra of ferrous and ferric cytochrome c peroxidase and Compound ES and their pH dependences were investigated in resonance with Soret band. The Fe(IV) = O stretching Raman line of Compound ES was assigned to a broad band around 767 cm-1, which was shifted to 727 cm-1 upon 18O substitution. The 18O-isotopic frequency shift was recognized for Compound ES derived in H218O, but not in H216O. This clearly indicated occurrence of an oxygen exchange between the Fe(IV) = O heme and bulk water. The Fe(IV) = O stretching Raman band was definitely more intense and of higher frequency in D2O than in H2O as in Compound II of horseradish peroxidase, but in contrast with this its frequency was unaltered between pH 4 and 11. The Fe(II)-histidine stretching Raman line was assigned on the basis of the frequency shift observed for 54Fe isotopic substitution. From the intensity analysis of this band, the pKa of the heme-linked ionization of ferrocytochrome c peroxidase was determined to be 7.3. The Raman spectrum of ferricytochrome c peroxidase strongly suggested that the heme is placed under an equilibrium between the 5- and 6-coordinate high-spin structures. At neutral pH it is biased to the 5-coordinate structure, but at the acidic side of the transition of pKa = 5.5 the 6-coordinate heme becomes dominant. F- was bound to the heme iron at pH 6, but Cl- was bound only at acidic pH. Acidification by HNO3, H2SO4, CH3COOH, HBr, or HI resulted in somewhat different populations of the 5- and 6-coordinate forms when they were compared at pH 4.3. Accordingly, it is inferred that a water molecule which is suggested to occupy the sixth coordination position of the heme iron is not coordinated to the heme iron at pH 6 but that protonation of the pKa = 5.5 residue induces an appreciable structural change, allowing the coordination of the water molecule to the heme iron.

Raman and infrared spectra of cytochrome c peroxidase-carbon monoxide adducts in alternative conformational states.

Resonance Raman (RR) spectra are reported for CO-bound cytochrome c peroxidase (CCP). At low pH, two forms are observed: form II, with nu Fe-C = 530 cm-1 and delta FeCO = 585 cm-1, and form I, with nu Fe-C = 495 cm-1 and no detectable delta FeCO. They appear to have coincident nu CO infrared bands, at 1922 cm-1. These low-pH forms, similar to those observed for horseradish peroxidase (HRP), are attributed to tilted, H-bonded CO and perpendicular CO, respectively. The frequencies differ between the two proteins, a weaker H bond to CO being indicated for CCP. As with HRP, the equilibrium between forms I and II is shifted toward the latter at increasing CO concentrations, suggesting that secondary binding of CO perturbs the distal residues. At high pH [8.4, tris(hydroxymethyl)aminomethane buffer] the form II fraction converts to another form, II', with nu FeC = 503 cm-1, delta FeCO = 575 cm-1, and nu CO = 1948 cm-1; a tilted, non-H-bonded geometry is suggested. If phosphate buffer is used, however, form II (H bonded) persists at pH 8.4. This result establishes a role for phosphate in stabilizing the H-bonded form of the enzyme; it is suggested that phosphate binds near the distal imidazole and substantially increases its pKa. The conformational state is also influenced by aging. Fresh protein contains purely high spin FeIII heme, as monitored by the high-frequency RR spectrum, and yields form II almost exclusively at elevated CO concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

The mechanism of the hormonal activation of respiration in isolated hepatocytes and its importance in the regulation of gluconeogenesis.

The effects of hormones on the cytochrome spectra of isolated hepatocytes were recorded under conditions of active gluconeogenesis from L-lactate. Glucagon, phenylephrine, vasopressin and valinomycin, at concentrations that caused stimulation of gluconeogenesis, increased the reduction of the components of the cytochrome bc1 complex, just as has been observed in liver mitochondria isolated from glucagon-treated rats [Halestrap (1982) Biochem. J. 204, 37-47]. The effects of glucagon and phenylephrine were additive. The time courses of the increased reduction of cytochrome c/c1 and NAD(P)H/NAD(P)+ caused by hormones, valinomycin, A23187 and ethanol were measured by dual-beam spectrophotometry and fluorescence respectively. Ethanol (14 mM) produced a substantial rise in NAD(P)H fluorescence, beta-hydroxybutyrate/acetoacetate and lactate/pyruvate ratios, no change in cytochrome c/c1 reduction, a 10% decrease in O2 consumption and a 60% decrease in gluconeogenesis. Glucagon, phenylephrine and vasopressin caused a substantial and transient rise in NAD(P)H fluorescence, but a sustained increase in cytochrome c/c1 reduction and the rates of O2 consumption and gluconeogenesis. The transience of the fluorescence response was greater in the absence of Ca2+, when the cytochrome c/c1 response also became transient. The fluorescence response was smaller and less transient, but the cytochrome c/c1 response was greater, in the presence of fatty acids. Both responses were greatly decreased by the presence of 1 mM-pent-4-enoate. Valinomycin (2.5 nM) caused a decrease in NAD(P)H fluorescence coincident with an increase in cytochrome c/c1 reduction and the rate of gluconeogenesis and O2 consumption. A23187 (7.5 mM) caused increases in both NAD(P)H fluorescence and cytochrome c/c1 reduction. The effects of hormones and valinomycin on the time courses of NAD(P)H fluorescence, cytochrome c/c1 reduction and light-scattering by hepatocytes were compared with those of 0.5 microM-Ca2+ or 1 nM-valinomycin on the same parameters of isolated liver mitochondria. It is concluded that hormones increase respiration by hepatocytes in a biphasic manner. An initial Ca2+-dependent activation of mitochondrial dehydrogenases rapidly increases the mitochondrial [NADH], which is followed by a volume-mediated stimulation of fatty acid oxidation and electron flow between NADH and cytochrome c. 10. Amytal (0.5 mM) was able to reverse the effects of hormones on the reduction of cytochromes c/c1 and the rates of gluconeogenesis and O2 consumption without significantly lowering tissue [ATP].(ABSTRACT TRUNCATED AT 400 WORDS)

Cytochrome c′-type cytochrome- c peroxidase derived from Nitrosomonas europaea

From Nitrosomonas europaea, a cytochrome c′-type haem protein has been purified which has cytochrome- c peroxidase (ferrocytochrome- c:hydrogen-peroxide oxidoreductase, EC 1.11.1.5) activity. The protein has haem c and shows the high-spin-type absorption spectra: it shows absorption peaks at 400, 500 and 640 nm in the oxidized form and peaks at 433 and 553 nm in the reduced form. Its spectra are changed into the haemochrome-type at pH higher than 11. The absorption spectra of the CO and NO complexes of its reduced form and the NO complex of its oxidized form resemble the respective spectra of cytochromes c′ from other bacteria. However, the haem protein has some characteristics which distinguish it from other cytochromes c′; (1) the protein has a cytochrome- c peroxidase activity, (2) its Soret band is composed of a single peak, (3) its oxidized form reacts with cyanide and azide easily, and (4) its molecule is composed of a haem-containing protein subunit (15 kDa) and a non-haem protein subunit (10 kDa). The molecular activities of the protein as cytochrome- c peroxidase are 1010 and 243 per min with horse and N. europaea cytochromes c, respectively.

Models for ferrous cytochrome b5: Sign inversions in the magnetic circular dichroism spectra of bis-imidazole ferrous porphyrin systems

The magnetic circular dichroism (MCD) spectrum of bis-imidazole ferrous tetraphenylporphyrin in the Soret region is nearly the mirror image of the spectrum of ferrous cytochrome b 5, a bis-imidazole (histidine)-ligated hemoprotein. Based on previous MCD studies of model and protein heme systems, a sign inversion in the spectra of two heme chromophores having essentially the same coordination structure is unexpected. To investigate whether the nature of the porphyrin itself could account for the observed spectral discrepancy, two additional model complexes, bis-imidazole ferrous protoporphyrin IX dimethylester and bis-imidazole ferrous octaethylporphyrin, whose peripheral porphyrin substituent patterns more closely match that of the protein- bound porphyrin, have been prepared and their MCD spectra measured. In these cases, the band pattern of the ferrous protein in the Soret region is successfully reproduced. It therefore appears that the anomalous MCD spectrum of the tetraphenylporphyrin complex can be attributed to the nature and positioning of the peripheral substituents on the porphyrin ring. Although iron tetraphenylporphyrin complexes are frequently used as models for protoporphyrin- containing hemoproteins, one should be aware that such differences in the peripheral porphyrin substituents may significantly affect the spectral properties of the model complex.

Difference spectroscopy with respiratory membranes of an H 2-oxidizing microorganism layered between two gas-permeable, plastic foils: A procedure that allows measurements in the ultraviolet range

A method for recording redox difference spectra of respiratory membranes which minimizes light scattering and results in increased sensitivity and spectral range when compared to the traditional technique of recording spectra of respiratory particles in suspension is described. Membranes were sandwiched between two gas-permeable, plastic foils, placed in a sealed cuvette, and gassed with H 2 as reductant or O 2 as oxidant. The membranes of the H 2-oxidizing microorganism ( Alcaligenes eutrophus H16) used in this work contain a hydrogenase which is coupled to the electron transport chain, thereby allowing H 2 to serve as a source of electrons. With this procedure, spectra of both cytochromes and quinones were recorded. By using mixtures of H 2 and O 2, spectra of cytochromes between the fully oxidized and fully reduced states were recorded.