Kinetics of oxidation of the bound cytochromes in reaction centers from Rhodopseudomonas viridis

The initial oxidized species in the photochemical charge separation in reaction centers from Rps. viridis is the primary donor, P⁺, a bacteriochlorophyll dimer. Bound c-type cytochromes, two high potential (Cyt c ₅₅₈) and two low potential (Cyt c ₅₅₃), act as secondary electron donors to P⁺. Flash induced absorption changes were measured at moderate redox potential, when the high potential cytochromes were chemically reduced. A fast absorption change was due to the initial oxidation of one of the Cyt c ₅₅₈ by P⁺ with a rate of 3.7×10⁶s^-1 (=270nsec). A slower absorption change was attributable to a transfer, or sharing, of the remaining electron from one high potential heme to the other, with a rate of 2.8×10⁵s^-1 (=3.5 sec). The slow change was measured at a number of wavelengths throughout the visible and near infrared and revealed that the two high potential cytochromes have slightly different differential absorption spectra, with -band maxima at 559 nm (Cyt c ₅₅₉) and 556.5 nm (Cyt c ₅₅₆), and dissimilar electrochromic effects on nearby pigments. The sequence of electron transfers, following a flash, is: Cyt c ₅₅₆Cyt c ₅₅₉P⁺. At lower redox potentials, a low midpoint potential cytochrome, Cyt c ₅₅₃, is preferentially oxidized by P⁺ with a rate of 7×10⁶s^-1 (=140 nsec). The assignment of the low and high potential cytochromes to the four, linearly arranged hemes of the reaction center is discussed. It is concluded that the closest heme to P must be the high potential Cyt c ₅₅₉, and it is suggested that a likely arrangement for the four hemes is: c ₅₅₃ c ₅₅₆ c ₅₅₃ c ₅₅₉P.Abbreviations diaminodurene 2,3,5,6-tetramethyl-p-phenylenediamine - MOPS 3-[N-morpholino]-propane-sulfonic acid - PMS methyl phenazinium methosulfate - PES ethyl phenazinium ethosulfate - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - TX-100 Triton X-100     

Biogenesis of mitochondrialc-type cytochromes

Cytochromesc andc ₁ are essential components of the mitochondrial respiratory chain. In both cytochromes the heme group is covalently linked to the polypeptide chain via thioether bridges. The location of the two cytochromes is in the intermembrane space; cytochromec is loosely attached to the surface of the inner mitochondrial membrane, whereas cytochromec ₁ is firmly anchored to the inner membrane. Both cytochromec andc ₁ are encoded by nuclear genes, translated on cytoplasmic ribosomes, and are transported into the mitochondria where they become covalently modified and assembled. Despite the many similarities, the import pathways of cytochromec andc ₁ are drastically different. Cytochromec ₁ is made as a precursor with a complex bipartite presequence. In a first step the precursor is directed across outer and inner membranes to the matrix compartment of the mitochondria where cleavage of the first part of the presequence takes place. In a following step the intermediate-size form is redirected across the inner membrane; heme addition then occurs on the surface of the inner membrane followed by the second processing reaction. The import pathway of cytochromec is exceptional in practically all aspects, in comparison with the general import pathway into mitochondria. Cytochromec is synthesized as apocytochromec without any additional sequence. It is translocated selectively across the outer membrane. Addition of the heme group, catalyzed by cytochromec heme lyase, is a requirement for transport. In summary, cytochromec ₁ import appears to follow a conservative pathway reflecting features of cytochromec ₁ sorting in prokaryotic cells. In contrast, cytochromec has invented a rather unique pathway which is essentially non-conservative.     

Characterization of the photosynthetic electron transport chain in normal and photobleachedAnabaena cylindrica by flash spectroscopy

Electron transport of normal and photobleachedAnabaena cylindrica was studied using spectral and kinetic analyses of absorbance transients induced by single turnover flashes. Between 500 and 600 nm two positive bands (540 and 566 nm) and two negative bands (515 and 554 nm) were found. Absorbance changes at 515 and 540 nm were partly characterized. None of these absorbance changes represent an electrochromic shift. Absorbance changes at 554 and 566 nm correspond to the oxidation of cytochromef and the reduction of cytochromeb ₅₆₃, respectively. We found a very slight 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) sensitivity of cytochromef in normal cells, while DCMU was completely ineffective for cytochromef reduction in photobleached cells. The absorbance change of cytochromeb ₅₆₃ increased, while the absorbance change of cytochromef was smaller than in normal cells. The increased O₂ evolution in photobleached cells and the negligible electron transport via cytochromef suggest the participation of other electron acceptor(s) in the electron-transport chain of photobleachedAnabaena cylindrica.     

C1 metabolism inParacoccus denitrificans: Genetics ofParacoccus denitrificans

Paracoccus denitrificans is able to grow on the C₁ compounds methanol and methylamine. These compounds are oxidized to formaldehyde which is subsequently oxidized via formate to carbon dioxide. Biomass is produced by carbon dioxide fixation via the ribulose biphosphate pathway. The first oxidation reaction is catalyzed by the enzymes methanol dehydrogenase and methylamine dehydrogenase, respectively. Both enzymes contain two different subunits in an ₂₂ configuration. The genes encoding the subunits of methanol dehydrogenase (moxF andmoxI) have been isolated and sequenced. They are located in one operon together with two other genes (moxJ andmoxG) in the gene ordermoxFJGI. The function of themoxJ gene product is not yet known.MoxG codes for a cytochromec _551i , which functions as the electron acceptor of methanol dehydrogenase. Both methanol dehydrogenase and methylamine dehydrogenase contain PQQ as a cofactor. These so-called quinoproteins are able to catalyze redox reactions by one-electron steps. The reaction mechanism of this oxidation will be described. Electrons from the oxidation reaction are donated to the electron transport chain at the level of cytochromec. P. denitrificans is able to synthesize at least 10 differentc-type cytochromes. Five could be detected in the periplasm and five have been found in the cytoplasmic membrane. The membrane-bound cytochromec ₁ and cytochromec ₅₅₂ and the periplasmic-located cytochromec ₅₅₀ are present under all tested growth conditions. The cytochromesc _551i andc _553i , present in the periplasm, are only induced in cells grown on methanol, methylamine, or choline. The otherc-type cytochromes are mainly detected either under oxygen limited conditions or under anaerobic conditions with nitrate as electron acceptor or under both conditions. An overview including the induction pattern of allP. denitrificans c-type cytochromes will be given. The genes encoding cytochromec ₁, cytochromec ₅₅₀, cytochromec _551i , and cytochromec _553i have been isolated and sequenced. By using site-directed mutagenesis these genes were mutated in the genome. The mutants thus obtained were used to study electron transport during growth on C₁ compounds. This electron transport has also been studied by determining electron transfer rates inin vitro experiments. The exact pathways, however, are not yet fully understood. Electrons from methanol dehydrogenase are donated to cytochromec _551i . Further electron transport is either via cytochromec ₅₅₀ or cytochromec _553i to cytochromeaa ₃. However, direct electron transport from cytochromec _551i to the terminal oxidase might be possible as well. Electrons from methylamine dehydrogenase are donated to amicyanin and then via cytochromec ₅₅₀ to cytochromeaa ₃, but other routes are used also.P. denitrificans is studied by several groups by using a genetic approach. Several genes have already been cloned and sequenced and a lot of mutants have been isolated. The development of a host/vector system and several techniques for mutation induction that are used inP. denitrificans genetics will be described.     

Cytochromebc purified from the methanogenMethanosarcina barkeri

Cytochromebc was partially purified from the methanogen,Methanosarcina barkeri. The cytochrome was composed of three subunits with molecular masses of 23.4, 20.9, and 9.1 kDa, respectively, and the 23.4 kDa subunit contained haemc. The absorption spectrum of cytochromebc showed a peak at 411 nm in the oxidized form, and peaks at 554, 524, and 422 nm in the reduced form. The cytochrome reacted with CO, and its low temperature absorption spectrum showed the peak at 552 nm with a shoulder at 557 nm.     

Interactions of cytochromec with phospholipid membranes

Summary Cytochromec added during the formation of lecithin-cardiolipin liquid crystals in 0.015m KCl is readily bound. After successive washings with 0.15m KCl, only about 50% of this bound cytochromec is removed. The remaining cytochromec is resistant to further salt extraction, and the amount of this cytochromec that is bound varies with the concentration of added cytochromec to a maximum binding ratio of 170, mole ratio cytochromec to phospholipid. This binding appears to be electrostatic; it is competitively inhibited by increasing the initial molarity of KCl from 0.015 to 0.10m. Binding of cytochromec is insignificant in the absence of cardiolipin, and is affected by varying the pH. Electron microscope studies of osmium tetroxide-stained thin sections show that the liquid crystals consist of vesicles, each of which contains a large number of concentric, alternating light and dense lines. The dense lines have been identified by other workers with the polar head groups of the phospholipids on the surface of a bilayer, and the light area represents the hydrophobic interior. The addition of cytochromec causes an average decrease in the number of lines per vesicle. It increases the center-to-center distance between two neighboring light or dense lines and the width of the dense lines. On the basis of this evidence and electrostatic binding, it is concluded that cytochromec is binding on the polar surfaces of the phospholipid bilayers comprising the liquid crystalline vesicles.     

Reducibility of cytochromeb in mitochondrial inner membrane

When mitochondrial inner membrane was disintegrated into Complex I-III, IV, and oligomycin-sensitive ATPase, about 50% of cytochromeb in Complex I-III was readily reduced with NADH, as judged by the appearance of a peak at 562 nm, while in whole mitochondria less than 25% of cytochromeb was reduced by succinate. On addition of antimycin to the substrate-reduced Complex I-III, cytochromeb was further reduced to 71% of the total, and the peak at 562 nm was red-shifted to 564 nm as in the case of dithionite reduction. These results indicate that the 562 nm and 564 nm peaks, at 29°C correspond, respectively, tob ₅₆₀ andb _562.5 at 77°K of Davis et al. [7] and to b _K and b _T of Chance et al. [2]. When Complex I-III and oligomycin-sensitive ATPase were reconstituted to form a membrane, about 60% of cytochromeb in Complex I-III was readily reduced with NADH. In this case the 562 nm peak was not red-shifted. However, the difference spectrum of NADH-reduced membraneminus that in the presence of deoxycholate showed a peak at 565 nm. A mirror image of the difference spectrum was obtained on addition of an uncoupler,m-chlorocarbonyl cyanide phenylhydrazone. This is characteristic for b _T. These results support the idea that the occurrence of spectral peaks of b _T and b _K is not due to two species but to single speciesAbbreviations OS-ATPase oligomycin sensitive ATPase - CCCP m-chlorocarbonyl cyanide phenylhydrazone - F₁ coupling factor one - OSCP oligomycinsensitivity-conferring protein     

b-Type cytochromes in plasma membranes ofPhaseolus vulgaris hypocotyls,Arabidopsis thaliana leaves, andZea mays roots

Summary The plasma membrane of higher plants contains more than one kind ofb-type cytochromes. One of these has a high redox potential and can be fully reduced by ascorbate. This component, the cytochromeb ₅₆₁ (cytb ₅₆₁), has its characteristic -band absorbance close to 561 nm wavelength at room temperature. Cytb ₅₆₁ was first isolated from etiolated bean hook plasma membranes by two consecutive anion exchange chromatography steps. During the first step performed at pH 8, cytb ₅₆₁ did not bind to the anion exchange column, but otherb-type cytochromes did. In the second step performed at pH 9.9, cytb ₅₆₁ was bound to the column and was eluted from the column at an ionic strength of about 100 mM KCl. However, when the same protocol was applied to the solubilized plasma membrane proteins fromArabidopsis thaliana leaves and maize roots, the ascorbate-reducible cytb ₅₆₁ bound already to the first anion exchange column at pH 8 and was eluted also at an ionic strength of about 100 mM KCl. Otherb-type cytochromes than the ascorbate-reducible cytb ₅₆₁ from the plasma membranes of Arabidopsis leaves and maize roots showed similar Chromatographic characteristics to that of bean hypocotyls. These results demonstrate particular differences in the Chromatographic behavior of cytb ₅₆₁ from different sources.Abbreviations cyt b ₅₆₁ cytochromeb ₅₆₁ - PM plasma membrane - PAGE polyacrylamide gel electrophoresis     

Purification and partial characterization of ad(−)-lactate dehydrogenase fromDesulfovibrio desulfuricans (ATCC 7757)

Summary A membrane-boundd(–)-lactate dehydrogenase (LDH), an important enzyme in carbon and energy metabolism in sulfate-reducing bacteria of the genusDesulfovibrio, was solubilized from the membrane fraction ofDesulfovibrio desulfuricans (ATCC 7757). The enzyme was purified 84-fold to a final specific activity of 525 nmol DCPIP-reduced/min/mg protein by ammonium sulfate precipitation, chloroform extraction, gel filtration with Sephadex G-150, and hydrophobic column chromatography withN-octylamine Sepharose 4B. The enzyme eluted off a Sephacryl S-300 column as a single peak with a molecular weight of 400 000±40 000 Da. Denaturing gel electrophoresis showed it to be composed of 5 protein bands. The oxidized and dithionite reduced spectra of LDH resembles the spectra ofc-type cytochromes found inDesulfovibrio species. The addition of lactate to LDH resulted in a partially reduced spectrum. The flavin/cytochromec/non-heme iron content per 400 000 Da LDH molecular weight was found to be 11.64.5. The LDH activity was specific ford(–)-lactate and had aK _m ford(–)-lactate of 4.3×10^–4 M. The pH optimum was between 6.5 and 8.5.     

Spectral characterization of c-type cytochromes purified from Beggiatoa alba

Three c-type cytochromes were purified from the filamentous sulfur-oxidizing bacterium, Beggiatoa alba strain B18LD, by ammonium sulfate fractionation, flat bed isoelectric focusing and gel filtration. Two of the cytochromes; flavocytochrome c-554 and cytochrome c, were similar to cytochromes found in anoxygenic photosynthetic bacteria. Flavocytochrome c-554 had an apparent molecular weight of 21,000, an isoelectric focusing point at pH 4.4, contained FMN as the flavin component and had absorption maxima at 410, 450 and 470 nm in the oxidized form and at 417, 523 and 554 nm in the dithionite-reduced from. Cytochrome c was also an acidic protein with a pI of 4.8 and an apparent molecular weight of 18,000. The absorption spectra maxima were at 400, 490 and 635 nm in the oxidized form, at 424 and 550 nm in the dithione-reduced form and at 415 and 555 nm in the dithionite-reduced plus CO form. The third cytochrome characterized, cytochrome c-553 had an apparent molecular weight of 13,000, an isoelectric point at pH 4.4 and showed absorption maxima at 411 nm in the oxidized form and at 418, 523 and 553 nm in the dithionite-reduced form. Cytochrome c-553 was also isolated as a complex with a non-heme protein with a molecular weight of 16,000. The non-heme protein altered the absorption spectra and isoelectric point of cytochrome c-553.Abbreviations IEF isoelectric focusing - M _r molecular weight - pI isoelectric point     

The role of the genesnrf EFG andccmFH in cytochromec biosynthesis inEscherichia coli

It has been suggested that two groups ofEscherichia coli genes, theccm genes located in the 47-min region and thenrfEFG genes in the 92-min region of the chromosome, are involved in cytochromec biosynthesis during anaerobic growth. The involvement of the products of these genes in cytochromec synthesis, assembly and secretion has now been investigated. Despite their similarity to other bacterial cytochromec assembly proteins, NrfE, F and G were found not to be required for the biosynthesis of any of thec-type cytochromes inE. coli. Furthermore, these proteins were not required for the secretion of the periplasmic cytochromes, cytochromec ₅₅₀ and cytochromec ₅₅₂, or for the correct targeting of the NapC and NrfB cytochromes to the cytoplasmic membrane. NrfE and NrfG are required for formate-dependent nitrite reduction (the Nrf pathway), which involves at least twoc-type cytochromes, cytochromec ₅₅₂ and NrfB, but NrfF is not essential for this pathway. Genes similar tonrfE, nrfF andnrfG are present in theE. coli nap-ccm locus at minute 47. CcmF is similar to NrfE, the N-terminal region of CcmH is similar to NrfF and the C-terminal portion of CcmH is similar to NrfG. In contrast to NrfF, the N-terminal, NrfF-like portion of CcmH is essential for the synthesis of allc-type cytochromes. Conversely, the NrfG-like C-terminal region of CcmH is not essential for cytochromec biosynthesis. The data are consistent with proposals from this and other laboratories that CcmF and CcmH form part of a haem lyase complex required to attach haemc to C-X-X-C-H haem-binding domains. In contrast, NrfE and NrfG are proposed to fulfill a more specialised role in the assembly of the formate-dependent nitrite reductase.     

The role ofc-type cytochromes in the terminal respiratory chain of the methylotrophic bacteriumMethylophilus methylotrophus

Whole cells of the methylotrophic bacteriumMethylophilus methylotrophus cultured under methanol-limited conditions contain approximately equal amounts of two majorc-type cytochromes,c _H andc _L. Virtually all of the cytochromec _H, and over one-third of the cytochromec _L, are loosely attached to the periplasmic surface of the respiratory membrane whence they can be released by sonication or by washing cells in ethylenediaminetetraacetate (EDTA). The latter causes inhibition of methanol oxidase activity and stimulation of ascorbate-N,N,N,N-tetramethyl-p-phenylenediamine (TMPD) oxidase activity, neither of which effects are reversible by divalent metal ions. Kinetic analyses indicate that ascorbate-TMPD is oxidised via two routes, viz. a slow low-affinity pathway involving loosely membrane-boundc-type cytochromes plus cytochrome oxidaseaa ₃, and a faster higher-affinity pathway involving the firmly membrane-bound cytochrome oxidasec _L o complex; the former route predominates in the presence of divalent metal ions, and the latter route after exposure to EDTA. These and other results are discussed in terms of the spatial organisation of the terminal respiratory chain, and of the role ofc-type cytochromes in the oxidation of methanol and ascorbate-TMPD.Abbreviations EDTA Enthylenediaminetetraacetate - PMS Phenazinemethosulphate - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - SDS Sodium dodecylsulphate - I₅₀ Concentration of inhibitor required to give 50% inhibition of enzyme activity - PQQ Pyrroloquinoline quinone     

Identification of an ascorbate-dependent cytochrome<Emphasis Type="Italic"> b</Emphasis> of the tonoplast membrane sharing biochemical features with members of the cytochrome<Emphasis Type="Italic"> b</Emphasis>561 family

Two membrane-bound, ascorbate-dependent b-type cytochromes were identified in etiolated bean (Phaseolus vulgaris L.) hypocotyls. Following solubilization of microsomal membranes and anion-exchange chromatography at pH 8.0, two major cytochrome peaks (P-I and P-II) were separated. Both cytochromes were reduced by ascorbate and re-oxidized by monodehydroascorbate, but P-I reduction by ascorbate was higher and saturated at far lower concentrations of ascorbate with respect to P-II. The -band was symmetrically centered at 561 nm in P-I, but it was asymmetric in P-II with a maximum at 562 nm and shoulder at 557 nm. Ascorbate reduction of P-II, but not P-I, was inhibited by diethyl pyrocarbonate. Reduced P-II but not P-I was readily oxidized by certain ferric chelates, including FeEDTA and Fe-nitrilotriacetic acid. Purified P-I, associated with the plasma membrane, showed up as a 63-kDa glycosylated protein during sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and behaved as a monomer of about 70 kDa during size-exclusion chromatography. P-I identified with a previously purified ascorbate-dependent b-type cytochrome of bean hypocotyl plasma membranes [P. Trost et al. (2000) Biochim Biophys Acta 1468:1–5]. Partially purified P-II, on the other hand, correlated with a heme-protein of 27 kDa in SDS–PAGE gels, was dimeric (60 kDa) during size-exclusion chromatography, and was associated with the tonoplast marker V-ATPase in sucrose gradients. The sequence of a peptide of 11 residues obtained by tryptic digestion of P-II was found to be identical to a segment of a putative cytochrome b561 of Zea mays and highly conserved in other related plant sequences, including that of Arabidopsis thaliana cytochrome b561-1 (CAA18169). The biochemical features fully support the assignment of P-II cytochrome to the family of cytochrome b561, ascorbate-dependent (CYBASC) cytochromes, which also includes cytochrome b561 of animal chromaffin granules. The presence of a cytochrome reducing ferric chelates on the tonoplast is consistent with the role of plant vacuoles in iron homeostasis.     

Effect of ligands on cytochromed fromAzotobacter vinelandii

Spectra of oxidized and reduced cytochromed in particles ofA. vinelandii were studied in the presence of the ligands CO, azide, and NH₂OH under oxidizing, reducing, and turnover conditions. Under oxidizing conditions, spectral changes were observed on oxidized cytochromed (absorption maximum at 648 nm) in the presence of CO and NH₂OH showing a shift of the maximum to shorter wavelengths (639 and 645 nm, respectively) and a broadening of the half-band width. Under reducing conditions, spectral changes were observed on reduced cytochromed (absorption maximum at 631 nm) in the presence of CO (absorption maximum at 636 nm), NO, NO^– ₂, and NH₂OH (absorption maximum at 642 nm in the presence of dithionite). The spectral changes of cytochromed in the presence of NH₂OH or with dithionite and NO^– ₂ were ascribed to the formation of the NO-cytochromed compound. Under turnover conditions CO, NH₂OH, and azide cause a spectral shift of the absorption maximum of cytochromed from 648 nm to 636, 645, and 655 nm, respectively. With NH₂OH and azide a broadening of the half-band width of 7 and 6 nm, respectively, was also observed. The spectral changes caused by CO and NH₂OH were interpreted as a binding of the ligands to cytochromed changing its conformation from the oxidized state absorbing at 648 nm into a more stable liganded form. Since azide does not affect the spectral bands of oxidized and reduced cytochromed, the spectral change during turnover in the presence of azide were ascribed to a preferential binding of azide to enzymically active conformation of cytochromed (cytochromed _x).     

A flash-photolysis study of the reactions of acaa 3-ttype cytochrome oxidase with dioxygen and carbon monoxide

The time course of absorbance changes following flash photolysis of the fully-reduced carboxycytochrome oxidase fromBacillus PS3 in the presence of O₂ has been followed at 445, 550, 605, and 830 nm, and the results have been compared with the corresponding changes in bovine cytochrome oxidase. The PS3 enzyme has a covalently bound cytochromec subunit and the fully-reduced species therefore accommodates five electrons instead of four as in the bovine enzyme. In the bovine enzyme, following CO dissociation, four phases were observed with time constants of about 10 s, 30 s, 100 s, and 1 ms at 445 nm. The initial, 10-s absorbance change at 445 nm is similar in the two enzymes. The subsequent phases involving hemea and Cu_A are not seen in the PS3 enzyme at 445 nm, because these redox centers are re-reduced by the covalently bound cytochromec, as indicated by absorbance changes at 550 nm. A reaction scheme consistent with the experimental observations is presented. In addition, internal electron-transfer reactions in the absence of O₂ were studied following flash-induced CO dissociation from the mixed-valence enzyme. Comparisons of the CO recombination rates in the mixed-valence and fully-reduced oxidases indicate that more electrons were transferred from hemea ₃ toa in PS3 oxidase compared to the bovine enzyme.     

Absorption and fluorescence spectra ofS-quinolylethylated Kunitz soybean trypsin inhibitor

Disulfide bonds in soybean trypsin inhibitor (Kunitz) were simultaneously reduced and alkylated using tri-n-butylphosphine and 2-vinylquinoline at pH 7.6 in 0.11 M Tris-4.4 M urea, 41% ethanol. The resulting S--2-quinolylethylated protein (2-QE-STI) has a new absorption peak at 315–318 nm. Its quinoline fluorescence can be excited above 310 nm independently of intrinsic protein fluorescence. Free 2-quinolylethylcysteine (2-QEC) shows unexpectedly weak fluorescence. Quinoline absorption in 2-QEC and 2-QE-STI changes with pH. The apparentpK values determined spectrophotometrically are near 5 for 2-QEC and 3 for 2-QE-STI. Fluorescence decreased with increasing pH and in the presence of chloride ions. Both structural and charge effects thus appear to influence the absorption and fluorescence of the quinoline group. Corrected fluorescence emission (excited at 316 nm) of neutral 2-QE-STI diluted in 0.1 N H₂SO₄ was directly proportional to concentration in the range 0.4–8 m 2-QEC. The 2-QEC content of the protein derivative determined by UV absorption at pH 1.5 was in agreement with the expected value of four residues per mole. Fluorescence measurements ofS-2-quinolylethylated proteins may be especially useful as a sensitive, specific assay for cyst(e)ine residues.Reference to a company or product name does not imply approval or recommendation of the product by the U.S. Department of Agriculture to the exclusion of others that may be suitable.Abbreviations used are Mops: 3-(N-morpholino)propanesulfonic acid; STI: soybean trypsin inhibitor (Kunitz); 2-PE-STI:S--2-pyridylethylated STI; 2-QEC:S--(2-quinolylethyl)-l-cysteine; 2-QE-STI:S--2-quinolylethylated STI; TosPheCH₂-trypsin: bovine trypsin treated withp-toluenesulfonyl phenylalanine chloromethyl ketone.     

Effects of iron-limitation ofEscherichia coli on growth,the respiratory chains and gallium uptake

The effects of iron limitation on growth, the composition and function of the respiratory chains, and gallium uptake inEscherichia coli have been studied. Decreasing the iron concentration in a defined medium using Chelex resin gave lower growth yields in both continuous culture and prolonged batch culture. In the former, ironlimited (entering [Fe]2.0 M) cells exhibited diminished respiration rates, respiration-driven proton translocation quotients, and levels of non-haem iron and cytochromes. The cellular concentration of haemoproteinb-590 (a cytochromea ₁-like hydroperoxidase) decreased 20-fold on iron limitation, whilst a CO-binding pigment with an absorption maximum in the dithionite-treated form near 500 nm appeared. Gallium(III) (9 M) added to iron-limited, but not iron-sufficient, cultures diminished growth yields further; cells grown with low entering concentrations of iron took up less gallium than iron-sufficient cells. These results are attributed to the interference by gallium(III) with siderophore-mediated metal uptake. Gallium also stimulated iron uptake and was itself accumulated by iron-sufficient cells, suggesting that gallium(III) also affects the iron transport system(s) of low affinity.     

G-Protein activation mediates prepulse facilitation of Ca2+ channel currents in bovine chromaffin cells

The effects of G-protein activation were investigated on tonic, large depolarization-induced Ca²⁺ channel facilitation in cultured bovine adrenal chromaffin cells. Under whole-cell voltage clamp, activation of G proteins by intracellular dialysis with 200 M GTP-S did not significantly affect prepulse facilitation or whole-cell Ba²⁺ current (I _Ba) density. In contrast, inactivation of G proteins by intracellular GDP-S or pertussis toxin (PTX) pretreatment completely abolished or markedly attenuated facilitation of I _Ba, respectively. GDP-S dialysis resulted in nearly a threefold increase in peak I _Ba density, whereas PTX pretreatment resulted in a 50% increase. Our results indicate that under control recording conditions (200 m intracellular GTP), G proteins are tonically activated and suppress high-voltage-activated (HVA) Ca²⁺ channels in a voltage-dependent and voltage-independent manner. Local superfusion of chromaffin cells with normal bath solution produced a rapid and reversible increase (50%) in I _Ba amplitudes that also abolished prepulse facilitation. Together, these results demonstrate that tonic facilitation of HVA Ca²⁺ channels in bovine chromaffin cells involves the voltage-dependent relief of a G-protein-mediated suppression, imposed by chromaffin cell secretory products that feedback and activate G-protein-coupled autoreceptors.This work was supported by a National Science Foundation grant (DCB-8812562), American Heart Association-Ohio Affiliate grant (SW-91-18), and an American Parkinson's Disease Association grant. C.A.D. was supported by a predoctoral National Research Service Award (National Institutes of Health training grant HL07571-08). The authors thank Kluener's Packing Co. for their generous supply of adrenal glands.     

Identification of b,c, and d cytochromes in the membrane of Vitreoscilla

Cytochromes b, c, d, and o were identified by spectroscopic analysis of respiratory membrane fragments from Vitreoscilla sp., strain C1. Carbon monoxide difference spectra of the reduced membranes had absorption maxima at 416, 534, and 571 nm (ascribed to cytochrome o) and 632 nm (cytochrome d). Derivative spectra of the pyridine hemochromogen spectra of the membranes identified the presence of b- and c-type cytochromes in Vitreoscilla. The cyanide binding curve of the membranes was biphasic with dissociation constants of 2.14 mM and 10.7 mM which were assigned to cytochrome o and cytochrome d, respectively. Membranes bound carbon monoxide with dissociation constant 3.9 M, which was assigned to cytochrome o. Cytochrome c ₅₅₆ and a NADH-p-iodonitrotetrazolium violet reductase component were partially purified from Vitreoscilla membranes.Abbreviations INT p-iodonitrotetrazolium violet - RMF respiratory membrane fragments - K _d dissociation constant - CHAPS 3-[(3-cholamido propyl) dimethylammonio]-1-propanesulfonate - DOC sodium deoxycholate - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate     

The effect of membrane potential on the redox state of cytochromeb 561 in antimycin-inhibited submitochondrial particles

The oxidation of cytochromeb ₅₆₁ by ATP was measured in submitochondrial particles inhibited by antimycin. The redox potential of the bulk (M phase) was controlled by the ratio of fumarate:succinate, and the oxidation of cytochromeb was calculated and expressed as a change in redox potential (E _h) measured in millivolts. The oxidation of cytochromeb ₅₆₁ is an energy-driven reaction affected only by the component of the proton motive force. The oxidation (measured in millivolts) is a function of the phosphate potential, reaching a maximal value of 40 mV at G_ATP<–12 kcal/mole. The maximal measured value of ATP-dependent was 100 mV. Thus only a fraction of the membrane potential effects the redox state of cytochromeb ₅₆₁. In contrast to the ATP-induced oxidation of cytochromeb ₅₆₁, cytochromeb ₅₆₆ is in redox equilibrium with fumarate succinate either in the presence or in the absence of ATP. The selective oxidation ofb ₅₆₁ is explained within the term of theQ cycle as a reflection of on the electron electrochemical potential. The positive electric potential of theC phase causes cytochromeb ₅₆₆ to act as oxidant with respect to cytochromeb ₅₆₁. In the presence of antimycin cytochromeb ₅₆₁ cannot equilibrate with the quinone and undergoes oxidation, while cytochromeb ₅₆₆ reequilibrates with the quinone and thus regains redox equilibrium with the fumarate succinate redox buffer.Abbreviations used: ETP_H, phosphorylating submitochondrial particles; TMPD,N ₁ N ₁ NN-tetramethyl-p-phenylenediamine; FCCP, carbonylcyanidep-trifluoromethoxyphenylhydrazone; Mes, 2-(N-morpholino) ethanesulfonic acid.