Low Midpoint Potential Research Articles

On the iron–sulfur clusters in the complex redox enzyme dihydropyrimidine dehydrogenase

Porcine liver dihydropyrimidine dehydrogenase is a homodimeric iron-sulfur flavoenzyme that catalyses the first and rate-limiting step of pyrimidine catabolism. The enzyme subunit contains 16 atoms each of nonheme iron and acid-labile sulfur, which are most likely arranged into four [4Fe-4S] clusters. However, the presence and role of such Fe-S clusters in dihydropyrimidine dehydrogenase is enigmatic, because they all appeared to be redox-inactive during absorbance-monitored titrations of the enzyme with its physiological substrates. In order to obtain evidence for the presence and properties of the postulated four [4Fe-4S] clusters of dihydropyrimidine dehydrogenase, a series of EPR-monitored redox titrations of the enzyme under a variety of conditions was carried out. No EPR-active species was present in the enzyme 'as isolated'. In full agreement with absorbance-monitored experiments, only a small amount of neutral flavin radical was detected when the enzyme was incubated with excess NADPH or dihydrouracil under anaerobic conditions. Reductive titrations of dihydropyrimidine dehydrogenase with dithionite at pH 9.5 and photochemical reduction at pH 7.5 and 9.5 in the presence of deazaflavin and EDTA led to the conclusion that the enzyme contains two [4Fe-4S]2+,1+ clusters, which both exhibit a midpoint potential of approximately -0.44 V (pH 9.5). The two clusters are most likely close in space, as demonstrated by the EPR signals which are consistent with dipolar interaction of two S = 1/2 species including a half-field signal around g approximately 3.9. Under no circumstances could the other two postulated Fe-S centres be detected by EPR spectroscopy. It is concluded that dihydropyrimidine dehydrogenase contains two [4Fe-4S] clusters, presumably determined by the C-terminal eight-iron ferredoxin-like module of the protein, whose participation in the enzyme-catalysed redox reaction is unlikely in light of the low midpoint potential measured. The presence of two additional [4Fe-4S] clusters in dihydropyrimidine dehydrogenase is proposed based on thorough chemical analyses on various batches of the enzyme and sequence analyses. The N-terminal region of dihydropyrimidine dehydrogenase is similar to the glutamate synthase beta subunit, which has been proposed to contain most, if not all, the cysteinyl ligands that participate in the formation of the [4Fe-4S] clusters of the glutamate synthase holoenzyme. It is proposed that the motif formed by the Cys residues at the N-terminus of the glutamate synthase beta subunit, which are conserved in dihydropyrimidine dehydrogenase and in several beta-subunit-like proteins or protein domains, corresponds to a novel fingerprint that allows the formation of [4Fe-4S] clusters of low to very low midpoint potential.

Primary structure of cytochrome c′ of Methylococcus capsulatus Bath: evidence of a phylogenetic link between P460 and c′-type cytochromes

Cytochrome c' of Methylococcus capsulatus Bath is involved in electron flow from the enzyme responsible for hydroxylamine oxidation, cytochrome P460, to cytochrome C555. This cytochrome is spectrally similar to other cytochromes c' but is larger (16,000 Da) and has a lower midpoint potential (-205 mV). By a combination of Edman degradation, mass spectroscopy, and gene sequencing, we have obtained the primary structure of cytochrome c' from M. capsulatus Bath. The cytochrome shows low sequence similarity to other cytochromes c', only residues R12, Y53, G56, and the C-terminal heme-binding region (GXXCXXCHXXXK) being conserved. In contrast, cytochrome c' from M. capsulatus Bath shows considerable sequence similarity to cytochromes P460 from M. capsulatus Bath (31% identity) and from Nitrosomonas europaea (18% identity). This suggests that P460-type cytochromes may have originated from a c'-type cytochrome which developed a covalent cross-link between a lysine residue and the c'-heme.

Periplasmic electron carriers and photo-induced electron transfer in the photosynthetic bacterium Ectothiorhodospira sp.

A detailed analysis of the periplasmic electron carriers of the photosynthetic bacterium Ectothiorhodospira sp. has been performed. Two low mid-point redox potential electron carriers, cytochrome c' and cytochrome c, are detected. A high potential iron-sulfur protein is the only high mid-point redox potential electron transfer component present in the periplasm. Analysis of light-induced absorption changes shows that this high potential iron-sulfur protein acts in vivo as efficient electron donor to the photo-oxidized high potential heme of the Ectothiorhodospira sp. reaction center.

A heme-C-containing enzyme complex that exhibits nitrate and nitrite reductase activity from the dissimilatory iron-reducing bacterium Geobacter metallireducens.

Nitrate reduction in the dissimilatory iron-reducing bacterium Geobacter metallireducens was investigated. Nitrate reductase and nitrite reductase activities in nitrate-grown cells were detected only in the membrane fraction. The apparent K(m )values for nitrate and nitrite were determined to be 32 and 10 microM, respectively. Growth on nitrate was not inhibited by either tungstate or molybdate at concentrations of 1 mM or less, but was inhibited by both at 10 and 20 mM. Nitrate and nitrite reductase activity in the membrane fraction was not, however, affected by dialysis with 20 mM tungstate. An enzyme complex that exhibited both nitrate and nitrite reductase activity was solubilized from membrane fractions with CHAPS and was partially purified by preparative gel electrophoresis. It was found to be composed of four different polypeptides with molecular masses of 62, 52, 36, and 16 kDa. The 62-kDa polypeptide [a low-midpoint potential (-207 mV), multiheme cytochrome c] exhibited nitrite reductase activity under denaturing conditions. No molybdenum was detected in the complex by plasma-emission mass spectrometry.

New insights into structure-function relationships in nitrogenase: a 1.6 Å resolution X-ray crystallographic study of Klebsiella pneumoniae MoFe-protein

The X-ray crystal structure of Klebsiella pneumoniae nitrogenase component 1 (Kp1) has been determined and refined to a resolution of 1.6 Å, the highest resolution reported for any nitrogenase structure. Models derived from three 1.6 Å resolution X-ray data sets are described; two represent distinct oxidation states, whilst the third appears to be a mixture of both oxidized and reduced states (or perhaps an intermediate state). The structures of the protein and the iron-molybdenum cofactor (FeMoco) appear to be largely unaffected by the redox status, although the movement of Ser β90 and a surface helix in the β subunit may be of functional significance. By contrast, the 8Fe-7S P-cluster undergoes discrete conformational changes involving the movement of two iron atoms. Comparisons with known component 1 structures reveal subtle differences in the FeMoco environment, which could account for the lower midpoint potential of this cluster in Kp1. Furthermore, a non-proline- cis peptide bond has been identified in the α subunit that may have a functional role. It is within 10 Å of the FeMoco and may have been overlooked in other component 1 models. Finally, metal-metal and metal-sulphur distances within the metal clusters agree well with values derived from EXAFS studies, although they are generally longer than the values reported for the closely related protein from Azotobacter vinelandii. A number of bonds between the clusters and their ligands are distinctly longer than the EXAFS values, in particular, those involving the molybdenum atom of the FeMoco.

A low-redox potential heme in the dinuclear center of bacterial nitric oxide reductase: implications for the evolution of energy-conserving heme-copper oxidases.

Bacterial nitric oxide reductase (NOR) catalyzes the two-electron reduction of nitric oxide to nitrous oxide. It is a highly diverged member of the superfamily of heme-copper oxidases. The main feature by which NOR is distinguished from the heme-copper oxidases is the elemental composition of the active site, a dinuclear center comprised of heme b(3) and non-heme iron (Fe(B)). The visible region electronic absorption spectrum of reduced NOR exhibits a maximum at 551 nm with a distinct shoulder at 560 nm; these are attributed to Fe(II) heme c (E(m) = 310 mV) and Fe(II) heme b (E(m) = 345 mV), respectively. The electronic absorption spectrum of oxidized NOR exhibits a characteristic shoulder around 595 nm that exhibits complex behavior in equilibrium redox titrations. The first phase of reduction is characterized by an apparent shift of the shoulder to 604 nm and a decrease in intensity. This is due to reduction of Fe(B) (E(m) = 320 mV), while the subsequent bleaching of the 604 nm band represents reduction of heme b(3) (E(m) = 60 mV). This separation of redox potentials (>200 mV) allows the enzyme to be poised in the three-electron reduced state for detailed spectroscopic examination of the Fe(III) heme b(3) center. The low midpoint potential of heme b(3) represents a thermodynamic barrier to the complete (two-electron) reduction of the dinuclear center. This may avoid formation of a stable Fe(II) heme b(3)-NO species during turnover, which may be an inhibited state of the enzyme. It would also appear that the evolution of significant oxygen reducing activity by heme-copper oxidases was not simply a matter of the substitution of copper for non-heme iron in the dinuclear center. Changes in the protein environment that modulate the midpoint redox potential of heme b(3) to facilitate both complete reduction of the dinuclear center (a prerequisite for oxygen binding) and rapid heme-heme electron transfer were also necessary.

Purification and properties of the heme- and iron-sulfur-containing heterodisulfide reductase from Methanosarcina thermophila.

The heterodisulfide reductase (HDR) from Methanosarcina thermophila was purified to homogeneity from acetate-grown cells. In the absence of detergents, HDR consisted of an eight-protein complex with hydrogenase activity. However, when HDR was purified in the presence of 0.6% Triton X-100, a two-subunit (53 and 27 kDa) high specific activity ( approximately 200 units mg-1) enzyme was obtained that lacked hydrogenase activity. Sedimentation equilibrium experiments demonstrated that HDR has a molecular mass of 206 kDa and a high partial specific volume (0.9 cm3/g), indicating that the purified protein contains a significant amount of bound lipid. Like the HDR from Methanosarcina barkeri [Kunkel, A., Vaupel, M., Heim, S., Thauer, R. K., and Hedderich, R. (1997) Eur. J. Biochem. 244, 226-234], it was found to contain two discrete b-type hemes in the small subunit and two distinct [Fe4S4]2+/1+ clusters in the large subunit. One heme is high-spin and has a high midpoint potential (-23 mV), whereas the other heme apparently is low-spin and exhibits a relatively low midpoint potential (-180 mV). Only the high-spin heme binds CO. The midpoint potentials for the two clusters are -100 and -400 mV. In the fully reduced state, a complicated EPR spectrum with g values of 2.03, 1.97, 1.92, and 1.88 was observed. This spectrum resembles that of 8Fe ferredoxins in the fully reduced state, indicating that the two clusters in HDR are near enough to experience relatively strong dipolar interactions. Kinetic studies in which CO oxidation is coupled to heterodisulfide reduction strongly indicate that a membrane-associated compound is the direct electron donor to HDR. An electron-transfer pathway is presented that postulates a mechanism for coupling electron transport to proton translocation.

Regulation of oxidation-reduction potentials through redox-linked ionization in the Y98H mutant of the Desulfovibrio vulgaris [Hildenborough] flavodoxin: direct proton nuclear magnetic resonance spectroscopic evidence for the redox-dependent shift in the pKa of Histidine-98.

Flavodoxin from Desulfovibrio vulgaris is a low molecular weight (15 000 Da) acidic flavoprotein that contains a single flavin mononucleotide (FMN) cofactor. A distinguishing feature of the flavodoxin family is the exceptionally low midpoint potential of the semiquinone/hydroquinone couple. Tyrosine-98, which flanks the outer or si face of the FMN, plays an important role in establishing the oxidation-reduction properties of the bound cofactor as demonstrated by the substitution of a number of amino acids at this position [Swenson, R. P., & Krey, G. D. (1994) Biochemistry 33, 8505-8514]. The midpoint potential for the semiquinone/hydroquinone couple increases substantially when basic residues are introduced at this position. The pH dependency in the Y98H mutant is consistent with a redox-linked ionization model in which the favorable electrostatic coupling between the imidazolium cation and the flavin hydroquinone anion is responsible for the higher potential. Such a model predicts an increase in the pKa of 1.5 units for His98 upon complete reduction of the FMN. In this study, proton nuclear magnetic resonance spectroscopy was used to directly determine the intrinsic pKa of His98 as a function of the redox state of the cofactor in this flavodoxin. Values for the pKa of His98 in the oxidized and fully reduced flavodoxin are 7.02 +/- 0.08 and 8.43 +/- 0.11, respectively, an increase in the pKa by 1.41 units, which conforms with the previous prediction. These results provide direct experimental proof of the redox-linked ionization of this residue and provides further evidence of the crucial role of electrostatic interactions, in this case, in the stabilization of the flavin hydroquinone anion. This phenomenon may represent a general mechanism in the modulation of the reduction potential of the flavin cofactor within flavoenzymes in which ionizable groups such as histidine in the active center change ionization states during the catalytic cycle.

Trans effects on cysteine ligation in the proximal His93Cys variant of horse heart myoglobin.

Three variants of horse heart myoglobin (Mb) in which the proximal His93 residue has been replaced with a Cys residue have been constructed and studied by NMR, EPR, and MCD spectroscopy to evaluate the contributions of proximal and distal residues to the coordination environment of the heme iron in these proteins. Although no experimental conditions were identified that allowed quantitative ligation of the cysteine residue to the heme iron in the His93Cys variant, all of the spectroscopic evidence collected for the His93Cys/His64Ile and His93Cys/His64Val double variants supports the assignment of thiolate as the ligand to iron in the oxidized forms of these variants. The double metMb variants exhibit Soret maxima that are considerably blue-shifted, 1H NMR spectra with decreased mean methyl resonances, and EPR spectra with highly rhombic g values. These spectroscopic data for the Fe(III) variants resemble the corresponding properties reported for ferricytochrome P-450. The decrease in the reduction potential of the double variants by 280 mV relative to wild-type protein is also consistent with the low midpoint potential of cytochrome P-450. MCD spectroscopy of these variants confirms that the proximal cysteine residue is not bound in the reduced forms of these proteins and, in the case of the His93Cys variant, that the distal histidine is coordinated to the iron. Similar coordination environments were created in the ferrimyoglobin variants by cyanogen bromide modification, which resulted in cyanation of the sulfur atom and prevented the ligation of Cys93 to the heme iron.(ABSTRACT TRUNCATED AT 250 WORDS)

Photometric characteristics of haem proteins in erythropoietin-producing hepatoma cells (HepG2).

Erythropoietin (Epo)-producing hepatoma cells (HepG2) reveal, in addition to the cytochromes of the respiratory chain, a photometrically measurable haem signal with absorbance maxima at 559 nm and 427 nm, suggesting the presence of a b-type cytochrome. This activity exhibited a low midpoint potential, CO-binding spectra and reduction which was insensitive to both cyanide and antimycin. This haem possessed a 22 kDa subunit and might be part of an electron transfer chain similar to the NADPH oxidase, since the NADPH oxidase cytosolic activating factor (p47) could be identified by Western blot analysis. H2O2, which was detected inside the cells by confocal microscopy, might therefore be produced by the suggested electron transfer chain. This cyanide- and antimycin-insensitive but hypoxia-sensitive cytochrome b would be an attractive candidate for controlled Epo production in response to pO2.

Nadph oxidase-like activity in rainbow trout Oncorhynchus mykiss (walbaum) macrophages

Evidence for the existence of an NADPH oxidase-like enzyme in rainbow trout macrophages is given. Reduced-minusoxidised difference spectroscopy revealed the presence of a cytochrome b with three absorbance peaks, at 430, 533, and 558 nm. The low midpoint potential of the latter peak suggests this cytochrome is the same as the terminal component of NADPH oxidase (i.e., cytochrome b −245). Subcellular fractionation of macrophages revealed two peaks of cytochrome b activity, in accord with the concept of a plasma membrane localisation of cytochrome b activity in addition to a mitochondrial localisation. Finally, that the rainbow trout oxidase is a multicomponent enzyme was suggested by inhibitor studies, where specific inhibitors of the flavin and cytochrome b −245 components of NADPH oxidase induced significant reduction in superoxide anion production.

Characterization of reaction center/antenna complexes from bacteriochlorophyll a containing Ectothiorhodospiraceae

Pigment-protein complexes have been isolated from three bacteriochlorophyll- a -containing Ectothiorhodospira species: E. mobilis, E. shaposhnikovii and E. halophila , using detergent solubilisation of photosynthetic membranes and sucrose-density gradient centrifugation. This isolation procedure yields pure LHII complexes and reaction centers with either one or both antenna complexes attached (i.e., RC/LHI and RC/LHI/LHII complexes). The RC/LHI/LHII complexes consist of three reaction center polypeptides, H (35 kDa), M (34 kDa), and L (26.5 kDa), plus two LHI and two LHII antenna polypeptides. In photosynthetic membranes of the three Ectothiorhodospira species two major types of heme c are present, of low (25 mV) and high (297 mV) midpoint potential, respectively. These heme groups belong to separate cytochromes. During solubilisation and purification, no loss of the H subunit from the reaction centers is observed. However, the reaction centers do lose their c -type cytochromes. The high-potential cytochrome c is completely lost during solubilisation of RC complexes from photosynthetic membranes, while the low-potential cytochrome is largely lost during isolation of RC/LHI complexes. The residual amount of this low-potential cytochrome, associated with the RC/LHI complexes, varies with the salt concentration used during the isolation, but remains below 0.2 mol heme c per mol reaction center. This is less than 20% of the amount originally present in photosynthetic membranes.

Redox regulation of the genes for cobinamide biosynthesis in Salmonella typhimurium.

Transcription of the cobinamide biosynthetic genes (the CobI operon) was induced under three different physiological conditions: anaerobiosis (anaerobic respiration or fermentation), aerobic respiration at low oxygen levels, and aerobic respiration with a partial block of the electron transport chain. After a shift to inducing conditions, there was a time lag of approximately 50 min before the onset of CobI induction. Under conditions of anaerobic respiration, the level of CobI transcription was dependent on the nature of both the electron donor (carbon and energy source) and the acceptor. Cells grown with electron acceptors with a lower midpoint potential showed higher CobI expression levels. The highest level of CobI transcription observed was obtained with glycerol as the carbon source and fumarate as the electron acceptor. The high induction seen with glycerol was reduced by mutational blocks in the glycerol catabolic pathway, suggesting that glycerol does not serve as a gratuitous inducer but must be metabolized to stimulate CobI transcription. In the presence of oxygen, CobI operon expression was induced 6- to 20-fold by the following: inhibition of cytochrome o oxidase with cyanide, mutational blockage of ubiquinone biosynthesis, and starvation of mutant cells for heme. We suggest that the CobI operon is induced in response to a reducing environment within the cell and not by the absence of oxygen per se.

The development of cytochrome b-245 in maturing human macrophages.

Cytochrome b-245, the putative terminal component of the specialized cidal oxidase system of phagocytes, was measured in human monocytes in culture. There was a dramatic synthesis of the cytochrome, which increased by 27.3 +/- 2.0 pmol/day per 10(7) cells. This represents an increase of about 40%/day in the early stages and an overall 7-fold increase after 16 days. The protein content increased 3-fold over the same period, resulting in a doubling of the specific content of the cytochrome b. The newly synthesized cytochrome b was identified as that specifically located in the microbicidal oxidase electron-transport chain, as titration demonstrated that, at day 16 of maturation, 70% of the total membrane cytochrome b had a very low midpoint potential (-260 to -220 mV), characteristic of that found in this oxidase system. This cytochrome distributed with the plasma membrane on analytical subcellular fractionation, and a close relationship was observed between the maturation-induced increase in the concentration of this molecule and the capacity of the cells to produce superoxide.

A study of the kinetic properties of the stable semiquinone of the reaction-center secondary acceptor in chromatophores of non-sulfur purple bacteria

Flash-induced absorption changes at 450 nm were investigated in isolated chromatophores of Rhodopseudomonas sphaeroides and Rhodospirillum rubrum non-sulfur purple bacteria to follow the redox changes of the semiquinone species of the secondary quinone acceptor of the photosynthetic reaction center. Excitation of a dark-adapted chromatophore suspension by a series of successive flashes in the presence of electron donors capable of rapidly reducing the photooxidized reaction-center pigment causes the formation of a stable semiquinone species (Q ⨪ B) with a lifetime which is shown to be proportional to the amount of the oxidized redox mediator in the incubation medium. It is shown that the disappearance of the flash-induced absorption changes at 450 nm on lowering the ambient redox potential ( E h) to 200–300 mV is the result of increasing the lifetime of Q ⨪ B, as the amount of the oxidized mediator diminishes; consequently, in these circumstances, the 2–5 min dark interval between the flash cycles appears insufficient for Q ⨪ B recovery. After the addition of redox mediators with a low midpoint potential, acting as an oxidant for Q ⨪ B, the flash-induced redox changes of Q ⨪ B were observed at low E h values unless E h reached a value at which Q B underwent reduction at equilibrium to form Q BH 2. The data provide evidence that reaction centers with a fully oxidized secondary acceptor can donate electrons to the cyclic electron-transport chain only after two turnovers, leading to the formation of the doubly reduced ubiquinone species (Q BH 2) of the secondary acceptor.

17] Respiratory burst during phagocytosis: An overview

This chapter provides an overview of the respiratory burst during phagocytosis. Granulocytes, some classes of macrophages, and natural killer cells release substantial amounts of superoxide (O 2 – ) and hydrogen peroxide (H 2 O 2 ) when their plasmalemma is perturbed. This response and the concomitant phenomena is termed as the “respiratory burst.” The perturbations induced by physiological processes can be mimicked by certain soluble or “quasi-soluble” agents. Moreover, superoxide and H 2 O 2 , formed on the external surface of the plasmalemma and internal surface of the phagosome, may interact to form hydroxyl radical and perhaps singlet oxygen. The most thoroughly studied cidal system known to function in the phagosome consists of myeloperoxidase, H 2 O 2 , and chloride. This system has been shown in vitro to be effective in killing bacteria, yeast, mycoplasma, viruses, and tumor cells. The complex nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity consists of at least two components—namely, a flavoprotein and a b -cytochrome with a low midpoint potential.

Flash-induced electron transport in b- and c-type cytochromes in Rhodospirillum rubrum. Evidence for a Q-cycle

Flash-induced electron flow in the cyclic chain in chromatophores of the purple photosynthetic bacterium Rhodospirillu rubrum was studied in order to obtain information about the reactions of various redox components and to test the validity of the Q-cycle model for electron transport. Redox reactions of the b-type cytochromes b-433 and b-428, the c-type cytochrome c-420 and the reaction center bacteriochlorophyll P-870 were observed at redox potentials between −50 and +350 mV in the presence and absence of the electron-transport inhibitors antimycin and n-heptadecylmercapto-6-hydroxy-5,8-quinolinequinone (HMHQQ). At all redox potentials, the reduction of the b-type cytochromes in the presence of antimycin was linked to the reduction of an equal amount of oxidized cytochrome c-420 or P-870 +. These results are interpreted as evidence for the functioning of a Q-cycle in the cyclic electron transport. The Q-cycle is inhibited by the quinone analogue HMHQQ. At potentials around 20 mV, in the absence of antimycin, cytochrome b-433 was reduced in a flash and subsequently oxidized with a simultaneous net oxidation of the dark-reduced cytochrome b-428 and reduction of the flash-oxidized cytochrome c-420. We conclude therefore that cytochrome b-433 has a lower midpoint potential than cytochrome b-428. Presumably, the midpoint potential of cytochrome b-428 is around 50 mV and the midpoint potential of cytochrome b-433 is below 0 mV. The concentration of the b-type cytochromes involved in light reactions was low. There seemed to be approximately one of each per six or eight reaction centers.

The b-type cytochromes of bovine heart mitochondria: Absorption spectra, enzymatic properties, and distribution in the electron transfer complexes

1. 1. Three b-type cytochromes ( b 557.5, b 560, and b 562.5), plus a chromophore with an absorption peak at 558 nm at 77 °K, have been found to be associated with the electron transport system of bovine heart mitochondria. The reduced minus oxidized spectra of these components at 77 °K, as well as that of cytochrome c 1, have been recorded with a wavelength accuracy of ± 0.1 nm and presented to the nearest 0.5 nm. All the major α and β absorption peaks of cytochromes b 557.5, b 560, b 562.5, c 1 and c have been shown by fourth derivative analysis to be present in the dithionite-reduced minus oxidized spectra of mitochondria and submitochondrial particles. 2. 2. The distribution of the above components has been studied in the four electron transfer complexes of the respiratory chain. Cytochromes b 560, b 562.5 and c 1, as well as chromophore-558, were found to fractionate into Complex III (reduced ubiquinone-cytochrome c reductase), whereas cytochrome b 557.5 was found in Complex II (succinate-ubiquinone reductase). 3. 3. Cytochrome b 560 was readily reduced by NADH or succinate, but b 562.5 was not reduced by substrates unless the preparation was treated with antimycin A. In antimycin-treated preparations pre-reduction of c 1 with ascorbate inhibited the subsequent reduction of b 562.5 by substrates. These results indicate that b 560 and b 562.5 correspond, respectively, to b K and b T previously described by Chance et al. 14 (1970, Proc. Natl. Acad. Sci. U.S. 66, 1175–1182). 4. 4. Similar to b 560, chromophore-558 can be reduced by substrates in the absence or presence of antimycin A. However, in antimycin-treated preparations, pre-reduction of c 1 inhibits its subsequent reduction by substrates. This property is similar to that of b 562.5. 5. 5. Cytochrome b 557.5, which occurs in Complex II, appears to have a low mid-point potential. It can be reduced with dithionite and oxidized by fumarate or ubiquinone. CO treatment of dithionite-reduced b 557.5 neither modified the spectrum of this cytochrome nor diminished the extent of b 557.5 reoxidation by fumarate. 6. 6. Antimycin A treatment does not appear to alter the spectra of the above cytochromes. However, small amounts (< 4%) of ethanol or methanol, which are usually added to particles as solvent for antimycin A, have a pronounced effect on the α peaks of cytochrome c 1. The spectrum of cytochrome c 1 at 77 °K as modified by 3% (v/v) ethanol is shown.

The redox state of cytochrome b559 in spinach chloroplasts

Spinach chloroplasts suspended in a mixture of redox buffers were titrated under anaerobic conditions by adding small amounts of oxidant (potassium ferricyanide) or reductant (reduced methyl viologen). At specific redox potentials, measured with a platinum electrode, samples of chloroplasts were removed, frozen to −196 °;C, and single-beam absorption spectra were measured before and after actinic irradiation at −196 °C. The extent of the low temperature light-induced reduction of C-550 and oxidation of cytochrome b 559 was determined as a function of redox potential. Difference spectra between samples at different redox potentials were used to determine titration curves for the chemically induced changes. With intact chloroplasts at 0 °C the midpoint potential of cytochrome b 559 was found to be + 450 mV. Titration of Tris-washed chloroplasts at 20 °C showed that cytochrome b 559 is oxidized at lower potentials and over a wider range of potentials than in intact chloroplasts. A cytochrome b 559 with a still lower midpoint potential (dithionite reducible but not ascorbate reducible) was also demonstrated in intact chloroplasts which had not been subjected to treatments which modify the normal high potential cytochrome b 559.

The respiratory chain of plant mitochondria XIII. Redox state changes of cytochrome b562 in mung bean seedling mitochondria treated with antimycin A

1. 1. The response to antimycin A of the redox state changes of cytochrome b 562, the cytochrome b in mung bean seedling mitochondria with long wavelength absorbance maximum and low midpoint potential of −77 mV independent of mitochondrial energy state, has been examined with regard to possible spectral shifts, with regard to the energy state of the mitchondria, and with regard to the redox state of the cytochromes c. 2. 2. Absorbance changes observed at 566 to 540 nm on addition of antimycin A to the mitochondrial suspension correspond primarily to changes in the redox state of cytochrome b 562. Spectral shifts contribute little or nothing to these changes. 3. 3. When a suspension of antimycin A-treated mung bean mitochondria becomes anaerobic with succinate as substrate through the alternate oxidase pathway characteristic of plant mitochondria, cytochrome b 562, which is highly reduced in the aerobic steady state, becomes partially oxidized. The degree of reoxidation is maximal in the presence of ATP and decreased by the presence of uncoupler or oligomycin. Reoxidation of the anaerobic mitochondrial suspension causes re-reduction of cytochrome b 562. 4. 4. The oxidation of cytochrome b 562 in antimycin A-treated mitochondria in the presence of ATP on transition to anaerobiosis is synchronous with the reduction of cytochrome c 549 (corresponding to c 1 in mammalian mitochondria) observed during this transition. Reduction of highly oxidized cytochrome c 549 in antimycin A-treated mitochondria by addition of sulfide during the aerobic steady state results in a corresponding oxidation of highly reduced cytochrome b 562. 5. 5. Oxidation of the b cytochromes by means of the alternate oxidase, which operates on the substrate side of these cytochromes with reference to cytochrome oxidase, is very slow in the presence of antimycin A when the c cytochromes are oxidized. 6. 6. It is concluded that b 562 and antimycin A form a stabilized complex analogous to the cytochrome a 3/CO complex, in which the reduced form is energetically favored when the c cytochromes are highly oxidized. The midpoint potential of the stabilized complex would be more positive than that of uncomplexed cytochrome b 562 by an amount determined by the free energies of formation of the antimycin A complex with oxidized and reduced b 562, as is the case with the CO complex of cytochrome a 3. When the c cytochromes become reduced, the stabilized cytochrome b 562/antimycin A complex relaxes, and in this form resumes a more negative midpoint potential. The cytochrome then is reoxidized by equilibration with oxidized carriers on its substrate side. It is proposed that this mechanism is also applicable to the changes in redox state of cytochrome b T observed in solubilized mitochondria preparations from heart muscle treated with antimycin A.