Initial purification study of the cytochromeb 561 of bean hypocotyl plasma membrane

Summary The plasma membrane (PM) of higher plants contains a major ascorbate-reducible, high-potentialb-type cytochrome, named cytochromeb ₅₆₁ (cytb ₅₆₁). In this paper a rapid purification protocol for the cytb ₅₆₁ of bean hypocotyls PM is described. An almost 200-fold increase of cytb ₅₆₁ specific concentration was achieved with respect to the PM fraction, which contained about 0.2 nmol of ascorbate-reducible heme per mg protein. The procedure can be performed in one day starting from purified PMs obtained by the phase-partitioning procedure. However, cytb ₅₆₁ proved to be unstable during chromatographic purification and the amount of protein finally recovered was low. Purified cytb ₅₆₁ eluted as a 130,000 Da protein-detergent complex from gel-filtration columns. It was completely reduced by ascorbate and reduced-minus-oxidized spectra showed -, - and -bands at 561, 530, and 429 nm respectively, not unlike the spectra of whole PMs. This work represents an initial approach to the biochemical characterization of the cytb ₅₆₁ of higher plants, formerly suggested to be related to cytb ₅₆₁ of animal chromaffin granules.Abbreviations cytb ₅₆₁ cytochromeb ₅₆₁ - PM plasma membrane - UPV upper-phase vesicles - GSII glucan synthase II - CCR NADH-dependent cytochromec reductase - CCO cytochromec oxidase - TX-100R reduced Triton X-100     

Organization and function of cytochromeb and ubiquinone in the cristae membrane of beef heart mitochondria

The arrangement and function of the redox centers of the mammalianbc ₁ complex is described on the basis of structural data derived from amino acid sequence studies and secondary structure predictions and on the basis of functional studies (i.e., EPR data, inhibitor studies, and kinetic experiments). Two ubiquinone reaction centers do exist—a QH₂ oxidation center situated at the outer, cytosolic surface of the cristae membrane (Q₀ center), and a Q reduction center (Q _i center) situated more to the inner surface of the cristae membrane. The Q₀ center is formed by theb-566 domain of cytochromeb, the FeS protein, and maybe an additional small subunit, whereas the Q _i center is formed by theb-562 domain of cytochromeb and presumably the 13.4kDa protein (QP-C). The Q binding proteins are proposed to be protein subunits of the Q reaction centers of various multiprotein complexes. The path of electron flow branches at the Q₀ center, half of the electrons flowing via the high-potential cytochrome chain to oxygen and half of the electrons cycling back into the Q pool via the cytochromeb path connecting the two Q reaction centers. During oxidation of QH₂, 2H⁺ are released to the cytosolic space and during reduction of Q, 2H⁺ are taken up from the matrix side, resulting in a net transport across the membrane of 2H⁺ per e^– flown from QH₂ to cytochromec, the H⁺ being transported across the membrane as H (H⁺ + e^–) by the mobile carrier Q. The authors correct their earlier view of cytochromeb functioning as a H⁺ pump, proposing that the redox-linkedpK changes of the acidic groups of cytochromeb are involved in the protonation/deprotonation processes taking place during the reduction and oxidation of Q. The reviewers stress that cytochromeb is in equilibrium with the Q pool via the Q _i center, but not via the Q₀ center. Their view of the mechanisms taking place at the reductase is a Q cycle linked to a Q-pool where cytochromeb is acting as an electron pump.     

A proposed pathway of proton translocation through thebc complexes of mitochondria and chloroplasts

The cytochromebc complexes of the electron transport chain from a wide variety of organisms generate an electrochemical proton gradient which is used for the synthesis of ATP. Proton translocation studies with radiolabeled N,N-dicyclohexylcarbodiimide (DCCD), the well-established carboxyl-modifying reagent, inhibited proton-translocation 50–70% with minimal effect on electron transfer in the cytochromebc ₁ and cytochromebf complexes reconstituted into liposomes. Subsequent binding studies with cytochromebc ₁ and cytochromebf complexes indicate that DCCD specifically binds to the subunitb and subunitb ₆, respectively, in a time and concentration dependent manner. Further analyses of the results with cyanogen bromide and protease digestion suggest that the probable site of DCCD binding is aspartate 160 of yeast cytochromeb and aspartate 155 or glutamate 166 of spinach cytochromeb ₆. Moreover, similar inhibition of proton translocating activity and binding to cytochromeb and cytochromeb ₆ were noticed with N-cyclo-N-(4-dimethylamino-napthyl)carbodiimide (NCD-4), a fluorescent analogue of DCCD. The spin-label quenching experiments provide further evidence that the binding site for NCD-4 on helix cd of both cytochromeb and cytochromeb ₆ is localized near the surface of the membrane but shielded from the external medium.     

Regulation of biosynthesis ofN-glycolylneuraminic acid-containing glycoconjugates: characterization of factors required for NADH-dependent cytidine 5′monophosphate-N-acetylneuraminic acid hydroxylation

The hydroxylation of CMP-NeuAc has been demonstrated to be carried out by several factors including the soluble form of cytochromeb ₅. In the present study, mouse liver cytosol was subjected to ammonium sulfate fractionation and cellulose phosphate column chromatography for the separation of two other essential fractions participating in the hydroxylation. One of the fractions, which bound to a cellulose phosphate column, was able to reduce the soluble cytochromeb ₅, using NADH as an electron donor. The other fraction, which flowed through the column, was assumed to contain the terminal enzyme which accepts electrons from cytochromeb ₅, activates oxygen, and catalyses the hydroxylation of CMP-NeuAc. Assay conditions for the quantitative determination of the terminal enzyme were established, and the activity of the enzyme in several tissues of mouse and rat was measured. The level of the terminal enzyme activity is associated with the expression ofN-glycolylneuraminic acid in these tissues, indicating that the expression of the terminal enzyme possibly regulates the overall velocity of CMP-NeuAc hydroxylation.Abbreviations CMP cytidine 5-monophosphate - NeuAc N-acetylneuraminic acid - NeuGc N-glycolylneuraminic acid - NADH reduced nicotinamide adenine dinucleotide - NADPH reduced nicotinamide adenine dinucleotide phosphate - DTT dithiothreitol     

Structural aspects of the cytochromeb 6 f complex; structure of the lumen-side domain of cytochromef

The following findings concerning the structure of the cytochromeb ₆ f complex and its component polypeptides, cytb ₆, subunit IV and cytochromef subunit are discussed:

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.     

Activation studies by phospholipids on the purified cytochromec 4:o oxidase ofAzotobacter vinelandii

A modified procedure is described that was used to solubilize and purify the TMPD-dependent cytochromec ₄:o oxidase fromAzotobacter vinelandii. Two functional components (Fractions I and V) were obtained after DEAE-cellulose chromatography. Fraction V contained both cytochromec ₄ (3.6 nmol/mg protein) and cytochromeo (1.6 nmol/mg protein). This cytochrome oxidase complex oxidized TMPD at moderate rates. Fraction I, a clear greenish-yellow fraction, contained primarily phosphatidylethanolamine with some phosphatidylglycerol. Fraction I itself could not oxidize TMPD, but when it was preincubated with Fraction V, a 2–4-fold stimulation in TMPD oxidase activity occurred. Other authentic micellar phospholipids also readily activited TMPD oxidase activity in Fraction V. Themaximum activation effect obtained with Fraction I was in essence duplicated with purified phosphatidylethanolamine.Dedicated to the memory of David E. Green, a fine gentleman, an excellent scientist, and a true scholar. He will be missed by many of his former colleagues.     

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     

A further investigation of the cytochrome<Emphasis Type="Italic"> b</Emphasis><Subscript>5</Subscript>–cytochrome<Emphasis Type="Italic"> c</Emphasis> complex

The interaction of reduced rabbit cytochrome b₅ with reduced yeast iso-1 cytochrome c has been studied through the analysis of ¹H–¹⁵N HSQC spectra, of ¹⁵N longitudinal (R₁) and transverse (R₂) relaxation rates, and of the solvent exchange rates of protein backbone amides. For the first time, the adduct has been investigated also from the cytochrome c side. The analysis of the NMR data was integrated with docking calculations. The result is that cytochrome b₅ has two negative patches capable of interacting with a single positive surface area of cytochrome c. At low protein concentrations and in equimolar mixture, two different 1:1 adducts are formed. At high concentration and/or with excess cytochrome c, a 2:1 adduct is formed. All the species are in fast exchange on the scale of differences in chemical shift. By comparison with literature data, it appears that the structure of one 1:1 adduct changes with the origin or primary sequence of cytochrome b₅.Electronic Supplementary Material Supplementary material is available for this article if you access the article at . A link in the frame on the left on that page takes you directly to the supplementary material.Abbreviations HSQC heteronuclear single quantum correlation spectroscopy - MD molecular dynamics     

Involvement of ascorbic acid and ab-type cytochrome in plant plasma membrane redox reactions

Summary Higher plant plasma membranes contain ab-type cytochrome that is rapidly reduced by ascorbic acid. The affinity towards ascorbate is 0.37 mM and is very similar to that of the chromaffin granule cytochromeb ₅₆₁. High levels of cytochromeb reduction are reached when ascorbic acid is added either on the cytoplasmic or cell wall side of purified plasma membrane vesicles. This result points to a transmembrane organisation of the heme protein or alternatively indicates the presence of an effective ascorbate transport system. Plasma membrane vesicles loaded by ascorbic acid are capable of reducing extravesicular ferricyanide. Addition of ascorbate oxidase or washing of the vesicles does not eliminate this reaction, indicating the involvement of the intravesicular electron donor. Absorbance changes of the cytochromeb -band suggest the electron transfer is mediated by this redox component. Electron transport to ferricyanide also results in the generation of a membrane potential gradient as was demonstrated by using the charge-sensitive optical probe oxonol VI. Addition of ascorbate oxidase and ascorbate to the vesicles loaded with ascorbate results in the oxidation and subsequent re-reduction of the cytochromeb. It is therefore suggested that ascorbate free radical (AFR) could potentially act as an electron acceptor to the cytochrome-mediated electron transport reaction. A working model on the action of the cytochrome as an electron carrier between cytoplasmic and apoplastic ascorbate is discussed.Abbreviations AFR ascorbate free radical - AO ascorbate oxidase - DTT dithiothreitol - FCCP carbonylcyanidep-trifluorome-thoxyphenylhydrazon - Hepes N-(2-hydroxyethyl)-piperazine-N-(2-ethanesulfonic acid) - Oxonol VI bis(3-propyl-5-oxoisoxazol-4-yl) penthamethine oxonol - PMSF phenylmethylsulfluoride     

Electron transfer from cytochromeb 5 to cytochromec

The reaction of cytochromeb ₅ with cytochromec has become a very prominent system for investigating fundamental questions regarding interprotein electron transfer. One of the first computer modeling studies of electron transfer and protein/protein interaction was reported using this system. Subsequently, numerous studies focused on the experimental determination of the features which control protein/protein interactions. Kinetic measurements of the intracomplex electron transfer reaction have only appeared in the last 10 years. The current review will provide a summary of the kinetic measurements and a critical assessment of the interpretation of these experiments.     

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.     

The reactions of the oxidase and reductases ofParacoccus denitrificans with cytochromesc

Electron transport in theParacoccus denitrificans respiratory chain system is considerably more rapid when it includes the membrane-bound cytochromec ₅₅₂ than with either solubleParacoccus c ₅₅₀ or bovine cytochromec; a pool function for cytochromec is not necessary. Low concentrations ofParacoccus or bovine cytochromec stimulate the oxidase activity. This observation could explain the multiphasic Scatchard plots which are obtained. A negatively charged area on the back side ofParacoccus c which is not present in mitochondrialc could be a control mechanism forParacoccus reactions.Paracoccus oxidase and reductase reactions with bovinec show the same properties as mammalian systems; and this is true ofParacoccus oxidase reactions with its own soluble cytochromec if added polycation masks the negatively charged area. Evidence for different oxidase and reductase reaction sites on cytochromec include: (1) stimulation of the oxidase but not reductase by a polycation; (2) differences in the inhibition of the oxidase and reductases by monoclonal antibodies toParacoccus cytochromec; and (3) reaction of another bacterial cytochromec withParacoccus reductases but not oxidase. Rapid electron transport occurs in cytochromec-less mutants ofParacoccus, suggesting that the reactions result from collision of diffusing complexes.     

Ubiquinol regeneration by plasma membrane ubiquinone reductase

Summary Several enzyme systems have been proposed to play a role in the maintenance of ubiquinol in membranes other than the inner mitochondrial membrane. The aim of this study was to investigate the mechanisms involved in NADH-driven regeneration of antioxidant ubiquinol at the plasma membrane. Regeneration was measured by quantifying the oxidized and reduced forms of ubiquinone by electrochemical detection after separation by high-performance liquid chromatography. Plasma membrane incubation with NADH resulted in the consumption of endogenous ubiquinone, and a parallel increase in ubiquinol levels. The activity showed saturation kinetics with respect to the pyridine nucleotides and was moderately inhibited byp-hydroxymercuribenzoate. Only a slight inhibition was achieved with dicumarol at concentrations reported to fully inhibit DT-diaphorase. Salt-extracted membranes displayed full activity of endogenous ubiquinol regeneration, supporting the participation of an integral membrane protein. In liposomes-reconstituted systems, the purified cytochromeb ₅ reductase catalyzed the reduction of the natural ubiquinone homologue coenzyme Q₁₀ at rates accounting for the activities observed in whole plasma membranes, and decreased the levels of lipid peroxidation. Our data demonstrate the role of the cytochromeb ₅ reductase in the regeneration of endogenous ubiquinol.Abbreviations AAPH 2,2-azobis-(2-amidinopropane) hydrochloride - CoQ coenzyme Q, ubiquinone - CoQH₂ reduced coenzyme Q, ubiquinol - pHMB p-hydroxymercuribenzoate     

Implications of cytochromeb 6/f location for thylakoidal electron transport

The cytochromeb ₆/f complex of higher plant chloroplasts is uniformly distributed throughout both appressed and nonappressed thylakoids, in contrast to photosystem II and photosystem I, the other major membrane protein complexes involved in electron transport. We discuss how this distribution is likely to affect interactions of the cytochromeb ₆/f complex with other electron transport components because of the resulting local stoichiometries, and how these may affect the regulation of electron transport.     

Effect of ubiquinone extraction on the reaction of the mitochondrialbc 1 complex with ferricyanide

Depletion of endogenous ubiquinone by pentane extraction of mitochondrial membranes lowered succinate-ferricyanide reductase activity, whereas quinone reincorporation restored the enzymatic activity as well as antimycin sensitivity. The oxidant-induced cytochromeb extrareduction, normally found upon ferricyanide pulse in intact mitochondria in the presence of antimycin, was lost in ubiquinone-depleted membranes, even if cytochromec was added. Readdition of ubiquinone-2 restored the oxidant-induced extrareduction with an apparent half saturation at 1 mol/molbc ₁ complex saturating at about 5 mol/mol. These findings demonstrate a requirement for the ubiquinone pool of the cytochromeb extrareduction. Since the initial rates of cytochromeb reoxidation upon ferricyanide addition, in the presence of antimycin, did not saturate by any ferricyanide concentration in ubiquinone-depleted mitochondria, a direct chemical reaction between ferricyanide and reduced cytochromeb was postulated. The fact that such direct reaction is much faster in ubiquinone-depleted mitochondria may explain the lower antimycin sensitivity of the succinate ferricyanide reductase activity after removal of endogenous ubiquinone.     

Mutational analysis of assembly and function of the iron-sulfur protein of the cytochromebc 1 complex inSaccharomyces cerevisiae

The iron-sulfur protein of the cytochromebc ₁ complex oxidizes ubiquinol at center P in the protonmotive Q cycle mechanism, transferring one electron to cytochromec ₁ and generating a low-potential ubisemiquinone anion which reduces the low-potential cytochromeb-566 heme group. In order to catalyze this divergent transfer of two reducing equivalents from ubiquinol, the iron-sulfur protein must be structurally integrated into the cytochromebc ₁ complex in a manner which facilitates electron transfer from the iron-sulfur cluster to cytochromec ₁ and generates a strongly reducing ubisemiquinone anion radical which is proximal to theb-566 heme group. This radical must also be sequestered from spurious reactivities with oxygen and other high-potential oxidants. Experimental approaches are described which are aimed at understanding how the iron-sulfur protein is inserted into center P, and how the iron-sulfur cluster is inserted into the apoprotein.     

Genomics of the<Emphasis Type="Italic"> ccoNOQP</Emphasis>-encoded<Emphasis Type="Italic"> cbb</Emphasis><Subscript>3</Subscript> oxidase complex in bacteria

Many bacteria adapt to microoxic conditions by synthesizing a particular cytochrome c oxidase (cbb ₃) complex with a high affinity for O₂, encoded by the ccoNOQP operon. A survey of genome databases indicates that ccoNOQP sequences are widespread in all sub-branches of Proteobacteria but otherwise are found only in bacteria of the CFB group (Cytophaga, Flexibacter, Bacteroides). Our analysis of available genome sequences suggests four major strategies of regulating ccoNOQP expression in response to O₂. The most widespread strategy involves direct regulation by the O₂-responsive protein Fnr. The second strategy involves an O₂-insensitive paralogue of Fnr, FixK, whose expression is regulated by the O₂-responding FixLJ two-component system. A third strategy of mixed regulation operates in bacteria carrying both fnr and fixLJ-fixK genes. Another, not yet identified, strategy is likely to operate in the -Proteobacteria Helicobacter pylori and Campylobacter jejuni which lack fnr and fixLJ-fixK genes. The FixLJ strategy appears specific for the -subclass of Proteobacteria but is not restricted to rhizobia in which it was originally discovered.     

Differential exposure of components of cytochromeb−c 1 region in beef heart mitochondria and electron transport particles

The reduction of cyctochromesc +c ₁ by durohydroquinone and ferrocyanide in electron transport particles (ETP) and intact cytochromec-depleted beef heart mitochondria has been studied. At least 94% of the ETP are in an inverted orientation. Durohydroquinone reduces 80% ofc +c ₁ in ETP but less than 20% in mitochondria; sonication of mitochondria allows reduction of cytochromesc +c ₁ (80%). Addition of ferrocyanide (effective redox potential +245 mV) to electron transport particles results in 30% reduction of cytochromesc +c ₁. Addition of ferrocyanide to intact cytochromec-depleted mitochondria does not reduce cytochromec ₁; treatment withN,N,N,N-tetramethylphenylenediamine, Triton X-100, or sonic oscillation results in 30% reduction of cytochromesc +c ₁. TheK _m value of ferrocyanide oxidase for K-ferrocyanide is pH-dependent in ETP only, increasing with increasing pH. The extent of reduction of cytochromec ₁ is also pH-dependent in ETP only, the extent of reduction increasing with decreasing pH. On the basis of these data cytochromec ₁ is exposed to the matrix face and cytochromec is exposed to the cytoplasmic face. No redox center other than cytochromec in the segment between the antimycin site and cytochromec is exposed on the C-side.Abbreviations Used: MES, 2(N-morpholino)-ethanesulfonic acid; EDTA, ethylenediaminetetraacetic acid; TMPD,N,N,N,N-tetramethylphenylenediamine; ETP, electron transport particles; NAD-NADH, nicotinamide adenine dinucleotide; PMS, phenazine methosulfate.