Membrane-bound cytochromes in Chloroflexus aurantiacus studied by EPR.

The heme components of chlorosome-depleted membranes of the green-gliding bacterium Chloroflexus aurantiacus were studied by EPR spectroscopy. The four major species, which are present in approximately equimolar quantities, are characterized by the following gz values, redox midpoint potentials and orientations of heme planes with respect to the plane of the membrane: gz = 3.40, Em = +280 mV, 30 degrees; gz = 3.33, Em = 0 mV, 45 degrees; gz = 3.03, Em = +95 mV, 40-50 degrees and gz = 2.95, Em = +150 mV, 90 degrees. These four hemes were attributed to cytochrome c554, the membrane-bound immediate electron donor to the photosynthetic reaction centre in Chloroflexus. All hemes except that with the highest potential were able to undergo photooxidation at 4 K. The photooxidation of the lowest potential heme was stable, whereas that of the +95 mV and the +150 mV hemes reversed on increasing the temperature to 100 K in darkness, due to charge recombination. The ability to photooxidize these hemes at 4 K was lost upon aging of samples. The results demonstrate that a reaction-centre-associated tetraheme cytochrome subunit, analogous to that of purple bacteria, is also present in C. aurantiacus.     

Electrochemical, FT-IR and UV/VIS spectroscopic properties of the caa3 oxidase from T. thermophilus.

The caa3-oxidase from Thermus thermophilus has been studied with a combined electrochemical, UV/VIS and Fourier-transform infrared (FT-IR) spectroscopic approach. In this oxidase the electron donor, cytochrome c, is covalently bound to subunit II of the cytochrome c oxidase. Oxidative electrochemical redox titrations in the visible spectral range yielded a midpoint potential of -0.01 +/- 0.01 V (vs. Ag/AgCl/3m KCl, 0.218 V vs. SHE') for the heme c. This potential differs for about 50 mV from the midpoint potential of isolated cytochrome c, indicating the possible shifts of the cytochrome c potential when bound to cytochrome c oxidase. For the signals where the hemes a and a3 contribute, three potentials, = -0.075 V +/- 0.01 V, Em2 = 0.04 V +/- 0.01 V and Em3 = 0.17 V +/- 0.02 V (0.133, 0.248 and 0.378 V vs. SHE', respectively) could be obtained. Potential titrations after addition of the inhibitor cyanide yielded a midpoint potential of -0.22 V +/- 0.01 V for heme a3-CN- and of Em2 = 0.00 V +/- 0.02 V and Em3 = 0.17 V +/- 0.02 V for heme a (-0.012 V, 0.208 V and 0.378 V vs. SHE', respectively). The three phases of the potential-dependent development of the difference signals can be attributed to the cooperativity between the hemes a, a3 and the CuB center, showing typical behavior for cytochrome c oxidases. A stronger cooperativity of CuB is discussed to reflect the modulation of the enzyme to the different key residues involved in proton pumping. We thus studied the FT-IR spectroscopic properties of this enzyme to identify alternative protonatable sites. The vibrational modes of a protonated aspartic or glutamic acid at 1714 cm-1 concomitant with the reduced form of the protein can be identified, a mode which is not present for other cytochrome c oxidases. Furthermore modes at positions characteristic for tyrosine vibrations have been identified. Electrochemically induced FT-IR difference spectra after inhibition of the sample with cyanide allows assigning the formyl signals upon characteristic shifts of the nu(C=O) modes, which reflect the high degree of similarity of heme a3 to other typical heme copper oxidases. A comparison with previously studied cytochrome c oxidases is presented and on this basis the contributions of the reorganization of the polypeptide backbone, of individual amino acids and of the hemes c, a and a3 upon electron transfer to/from the redox active centers discussed.     

On the question of interheme electron transfer in the chloroplast cytochrome b6 in situ

The redox properties, the site of action of the inhibitor NQNO, and the question of interheme transfer in the chloroplast cytochrome b6 have been examined with regard to the role of the b6-f complex in quinol oxidation and H+ translocation. (i) The two hemes of the cytochrome ba and bp, have similar (delta Em less than or equal to 50 mV) oxidation-reduction midpoint potentials that are pH-independent in the range pH 6.5-8.0 (Em7 = -40 mV) but are pH dependent below this range with an estimated pK = 6.7. (ii) Only half of cytochrome b6, the stromal-side heme, ba, was reducible by NADPH and ferredoxin. (iii) The 2-3-fold increase (to 0.60 +/- 0.09 heme/600 Chl) in the amplitude of flash-induced cytochrome reduction caused by NQNO was not affected when heme ba was initially reduced, implying that NQNO affects flash reduction at the site of heme bp. (iv) Multiple light flashes did not increase the amplitude of b6 reduction in the presence or absence of NQNO or show binary oscillations. Together with localization of a site of action of NQNO near heme bp, these data provide no evidence for efficient electron transfer from heme bp to heme ba as specified by the Q cycle model. (v) NQNO interaction with heme bp does not block its oxidation, since reoxidation of the flash-reduced cytochrome in its presence or absence was 4-5 times faster (t1/2 approximately 30 ms) when heme ba was reduced. The faster oxidation of the photoreduced cytochrome after NADPH-Fd reduction of heme ba indicates that the oxidation of ba and bp may be cooperative.     

Photo-induced cyclic electron transfer involving cytochrome bc1 complex and reaction center in the obligate aerobic phototroph Roseobacter denitrificans.

Flash-induced redox changes of b-type and c-type cytochromes have been studied in chromatophores from the aerobic photosynthetic bacterium Roseobacter denitrificans under redox-controlled conditions. The flash-oxidized primary donor P+ of the reaction center (RC) is rapidly re-reduced by heme H1 (Em,7 = 290 mV), heme H2 (Em,7 = 240 mV) or low-potential hemes L1/L2 (Em,7 = 90 mV) of the RC-bound tetraheme, depending on their redox state before photoexcitation. By titrating the extent of flash-induced low-potential heme oxidation, a midpoint potential equal to -50 mV has been determined for the primary quinone acceptor QA. Only the photo-oxidized heme H2 is re-reduced in tens of milliseconds, in a reaction sensitive to inhibitors of the bc1 complex, leading to the concomitant oxidation of a cytochrome c spectrally distinct from the RC-bound hemes. This reaction involves cytochrome c551 in a diffusional process. Participation of the bc1 complex in a cyclic electron transfer chain has been demonstrated by detection of flash-induced reduction of cytochrome b561, stimulated by antimycin and inhibited by myxothiazol. Cytochrome b561, reduced upon flash excitation, is re-oxidized slowly even in the absence of antimycin. The rate of reduction of cytochrome b561 in the presence of antimycin increases upon lowering the ambient redox potential, most likely reflecting the progressive prereduction of the ubiquinone pool. Chromatophores contain approximately 20 ubiquinone-10 molecules per RC. At the optimal redox poise, approximately 0.3 cytochrome b molecules per RC are reduced following flash excitation. Cytochrome b reduction titrates out at Eh < 100 mV, when low-potential heme(s) rapidly re-reduce P+ preventing cyclic electron transfer. Results can be rationalized in the framework of a Q-cycle-type model.     

Electrochemical, FTIR, and UV/VIS spectroscopic properties of the ba(3) oxidase from Thermus thermophilus.

The ba3 cytochrome c oxidase from Thermus thermophilus has been studied with a combined electrochemical, UV/VIS, and FTIR spectroscopic approach. Oxidative electrochemical redox titrations yielded midpoint potentials of Em1= -0.02 +/- 0.01 V and Em2 = 0.16 +/- 0.04 V for heme b and Em1 = 0.13 +/- 0.04 V and Em2 = 0.22 +/- 0.03 V for heme a(3) (vs Ag/AgCl/3 M KCl). Fully reversible electrochemically induced UV/VIS and FTIR difference spectra were obtained for the full potential step from -0. 5 to 0.5 V as well as for the critical potential steps from -0.5 to 0.1 V (heme b is fully oxidized and heme a3 remains essentially reduced) and from 0.1 to 0.5 V (heme b remains oxidized and heme a3 becomes oxidized). The difference spectra thus allow to us distinguish modes coupled to heme b and heme a3. Analogous difference spectra were obtained for the enzyme in D2O buffer for additional assignments. The FTIR difference spectra reveal the reorganization of the polypeptide backbone, perturbations of single amino acids and of hemes b and a3 upon electron transfer to/from the four redox-active centers heme b and a3, as well as CuB and CuA. Proton transfer coupled to redox transitions can be expected to manifest in the spectra. Tentative assignments of heme vibrational modes, of individual amino acids, and of secondary structure elements are presented. Aspects of the uncommon electrochemical and spectroscopic properties of the ba3 oxidase from T. thermophilus are discussed.     

Electrochemical titration of the cytochrome hemes in the Rhodopseudomonas viridis reaction center. Cyclic equilibrium titrations yield midpoint potentials without evidence for heme cooperativity.

The redox and spectral characteristics of the 4-heme cytochrome c unit of the photochemical reaction center from Rhodopseudomonas viridis were studied by a combination of protein electrochemistry and spectroscopy using an ultra thin-layer spectroelectrochemical cell. Quantitative and reversible reduction of the high-potential and the low-potential hemes was performed in cyclic titrations to record the optical difference spectra in the alpha-band region. The titration of the absorbance from the high-potential hemes can be approximated with a sum of 2 Nernst functions with Em = 0.113 V and Em = 0.175 V. The corresponding titration of the absorbance from the low-potential hemes yielded Em = -0.257 V and Em = -0.175 V (all potentials quoted vs. Ag/AgC1/3 M KCl; add 0.208 V for potentials vs. standard hydrogen electrode). The high-potential hemes equilibrate rapidly and titrate identically for oxidative and reductive titrations. Under identical conditions, the low-potential hemes exhibit a hysteresis, thus indicating much slower equilibration with the applied potential. Cyclic titrations with increasing equilibration periods, however, indicate the disappearance of the hysteresis for equilibration periods approximately twice as long as for the high-potential hemes. We take this as evidence for a slower internal equilibration, but against a cooperativity of the low-potential hemes as observed for other multi-heme cytochromes.     

Heme-copper and heme-heme interactions in the cytochrome bo-containing quinol oxidase of Escherichia coli

The cytochrome bo quinol oxidase of Escherichia coli is one of two respiratory O2 reductases which the bacterium synthesizes. The enzyme complex contains copper and 2 mol of b-type heme. Electron paramagnetic resonance (epr) spectroscopy of membranes from a strain having amplified levels of this enzyme complex reveals signals from low- and high-spin b-type hemes, but the copper, now established as a component of the oxidase, is not directly detectable by epr. The high-spin signal from the cytochrome bo complex, which we attribute to cytochrome o, when titrated potentiometrically, gives a bell-shaped curve. The low potential side of this curve is biphasic (Em7 approximately 180 and 280 mV) and corresponds to the reduction/oxidation of the cytochrome(s). The high potential side of the bell-shaped curve is monophasic (Em7 approximately 370 mV) and is proposed to be due to reduction/oxidation of a copper center which, when in the Cu(II) form, is tightly spin-coupled to a heme, probably cytochrome o, resulting in a net even spin system and loss of the epr spectrum. The low-spin cytochrome b titrates biphasically with Em7 values of approximately 180 and 280 mV, similar to the high-spin component but without the loss of signal at high potentials.     

Mitochondrial respiratory chain of Tetrahymena pyriformis: the properties of submitochondrial particles and the soluble b and c type pigments.

Submitochondrial particles isolated from Tetrahymena pyriformis contain essentially the same redox carriers as those present in parental mitochondria: at pH 7.2 and 22 degree C there are two b-type pigments with half-reduction potentials of --0.04 and --0.17 V, a c-type cytochrome with a half reduction potential of 0.215 V, and a two-component cytochrome a2 with Em7.2 of 0.245 and 0.345 V. EPR spectra of the aerobic submitochondrial particles in the absence of substrate show the presence of low spine ferric hemes with g values at 3.4 and 3.0, a high spin ferric heme with g =6, and a g=2.0 signal characteristic of oxidized copper. In the reduced submitochondrial particles signals of various iron-sulfur centers are observed. Cytochrome c553 is lost from mitochondria during preparation of the submitochondrial particles. The partially purified cytochrome c553 is a negatively charged protein at neutral pH with an Em7.2 of 0.25 V which binds to the cytochrome c-depleted Tetrahymena mitochondria in the amount of 0.5 nmol/mg protein with KD of 0.8.10(-6) M. Reduced cytochrome c553 serves as an efficient substrate in the reaction with its own oxidase. The EPR spectrum of the partially purified cytochrome c553 shows the presence of a low spin ferric heme with the dominant resonance signal at g=3.28. A pigment with an alpha absorption maximum at 560 nm can be solubilized from the Tetrahymena cells with butanol. This pigments has a molecular weight of approx. 18 000, and Em7.2 of--0.17 V and exhibits a high spin ferric heme signal at g=6.     

Electrogenic steps in the redox reactions catalyzed by photosynthetic reaction-centre complex from Rhodopseudomonas viridis

Electrogenic and redox events in the reaction-centre complexes from Rhodopseudomonas viridis have been studied. In contrast to the previous points of view it is shown that all the four hemes of the tightly bound cytochrome c have different Em values (-60, +20, +310 and +380 mV). The first three hemes reveal alpha absorption maxima at 554 nm, 552 nm and 556 nm respectively. The 380-mV heme displays a split alpha band with a maximum at 559 nm and a shoulder at 552 nm. Such a splitting is due to non-degenerated Qx and Qy transitions in the iron-porphyrin ring as demonstrated by magnetic circular dichroism spectra. Fast kinetic measurements show that, at redox potentials when only high-potential hemes c-559 and c-556 are reduced, heme c-559 appears to be the electron donor to P-960+ (tau = 0.32 microsecond) whereas heme c-556 serves to rereduce c-559 (tau = 2.5 microsecond). Upon reduction of the third heme (c-552), the P-960+ reduction rate increases twofold (tau = 0.17 microsecond) and all photoinduced redox events within the cytochrome appear to be complete in less than 1 microsecond after the flash. The following sequence of the redox centers is tentatively suggested: c-554, c-556, c-552, c-559, P-960. To study electrogenesis, the reaction-centre complexes from Rps. viridis were incorporated into asolectin liposomes, and fast kinetics of laser flash-induced electric potential difference has been measured in proteoliposomes adsorbed on a phospholipid-impregnated film. The electrical difference induced by a single 15-ns flash was found to be as high as 100 mV. The photoelectric response has been found to involve four electrogenic stages associated with (I) QA reduction by P-960; (II) reduction of P-960+ by heme c-559; (III) reduction of c-559 by c-556 and (IV) protonation of Q2-B. The relative contributions of stages I, II, III and IV are found to be equal to 70%, 15%, 5% and 10%, respectively, of the overall electrogenic process. At the same time, the first three respective distances along the axis normal to the membrane plane covered by electrons, calculated from X-ray data of Deisenhofer et al. [J. Mol. Biol. 180, 385-398 (1984)], are 22%, 18.5% and 26%. This indicates that the efficiency of electrogenic phases depends first of all upon the value of the dielectric constant of the respective membrane regions rather than upon the distance between the redox groups involved.(ABSTRACT TRUNCATED AT 400 WORDS)     

Respiratory electron transport and light-induced energy transduction in membranes from the aerobic photosynthetic bacterium Roseobacter denitrificans

Membrane fragments isolated from the aerobic phototrophic bacterium Roseobacter denitrificans were examined. Ninety-five percent of the total NADH-dependent oxidative activity was inhibited either by antimycin A or myxothiazol, two specific inhibitors of the cytochrome bc1 complex, which indicates that the respiratory electron transport chain is linear. In agreement with this finding, light-induced oxygen uptake, an electron transport activity catalyzed by the "alternative quinol oxidase pathway" in membranes of several facultative phototrophic species, was barely detectable in membranes of Rsb. denitrificans. Redox titrations at 561-575 nm, 552-540 nm, and 602-630 nm indicated the presence of three b-type cytochromes (Em,7 of +244 +/- 8, +24 +/- 3, -163 +/- 11 mV), four c-type cytochromes (Em,7 of +280 +/- 10, +210 +/- 5, +125 +/- 8, and 20 +/- 3 mV) and two a-type cytochromes (Em,7 of +335 +/- 15, +218 +/- 18 mV). The latter two a-type hemes were shown to be involved in cytochrome c oxidase activity, which was inhibited by both cyanide (I50 = 2 microM) and azide (I50 = 1 mM), while a soluble cytochrome c (c551, Em,7 = +217 +/- 2 mV) was shown to be the physiological electron carrier connecting the bc1 complex to the cytochrome c oxidase. A comparison of the ATP synthesis generated by continuous light in membranes of Rsb. denitrificans and Rhodobacter capsulatus showed that in both bacterial species photophosphorylation requires a membrane redox poise at the equilibrium (Eh > or = +80 < or = +140 mV), close to the oxidation-reduction potential of the ubiquinone pool. These data, taken together, suggest that, although the photosynthetic apparatus of Rsb. denitrificans is functionally similar to that of typical anoxygenic phototrophs, e.g. Rba. capsulatus, the in vivo requirement of a suitable redox state at the ubiquinone pool level restricts the growth capacity of Rsb. denitrificans to oxic conditions.     

Human neutrophil cytochrome b contains multiple hemes. Evidence for heme associated with both subunits.

Human phagocyte cytochrome b is the terminal component of the microbicidal superoxide generating system. Although the primary structure of this protein has been determined, little is known about the placement of the heme prosthetic groups in this heterodimeric integral membrane protein. Analysis of the cytochrome using lithium dodecyl sulfate-polyacrylamide gel electrophoresis at 0 degree C followed by tetramethylbenzidine heme staining demonstrated the presence of heme in both the 91- and 22-kDa subunits identified by Western blot analysis using peptide specific antisera. Exposure of cytochrome b (purified or in isolated neutrophil plasma membranes) to Staphylococcal protease V8 or trypsin did not affect absorbance spectra. However, such treatment resulted in degradation of both subunits to smaller fragments, including characteristic immunoreactive 20-kDa fragments of both the large and small subunits of the cytochrome that retained one or both of the hemes. The spectral stability to proteolysis and size of the proteolytic heme-containing fragments generated explains previous reports which suggested that the heme resided in the small subunit. Our current results indicate that human neutrophil cytochrome b is a bi-heme or possibly tri-heme molecule with at least one heme residing in the large subunit and one shared between both subunits and that the heme-containing regions of the cytochrome probably lie within the membrane lipid bilayer. Such a multi-heme structure would be consistent with an electron transfer function for this cytochrome by providing an efficient mechanism for transferring electrons across the plasma membrane to the extracellular surface where oxygen could be reduced to create superoxide.     

In vitro kinetics of reduction of cytochrome c554 isolated from the reaction center of the green phototrophic bacterium, Chloroflexus aurantiacus

The photochemical reaction center in the green bacterium Chloroflexus aurantiacus is similar to that found in purple phototrophic bacteria and interacts with a multiheme membrane-bound cytochrome. We have examined the kinetics of reduction of the pure solubilized reaction center cytochrome by laser flash photolysis of solutions containing lumiflavin or FMN. Reduction by lumiflavin semiquinone followed single exponential kinetics and the observed rate constant (kobs) was linearly dependent on protein concentration (k = 1.8 X 10(7) M-1s-1 heme-1). This result suggests either that the four hemes have similar reduction rate constants which cannot be resolved or that there are large differences in rate constant and only the most reactive heme (or hemes) was observed under these conditions. To determine the relative reactivities of the four hemes, we varied the extent of heme reduction at a single total protein concentration. As the hemes were progressively reduced by steady-state illumination prior to laser flash photolysis, kobs for the reaction with fully reduced lumiflavin decreased nonlinearly. Second-order rate constants for the four hemes were assigned by nonlinear least-squares analysis of kobs vs oxidized heme concentration data. The second-order rate constants obtained in this way for the highest and lowest potential hemes differed by a factor of about 20, which is larger than expected for c-type cytochromes based on redox potential alone (a factor of about 3 would be expected). This is interpreted as being due to differences in steric accessibility. Relative to the highest potential heme, which is as reactive as a typical c-type cytochrome, we estimated a steric effect of approximately twofold for heme 2, and steric effects of approximately fivefold for hemes 3 and 4. Using fully reduced FMN as reductant of oxidized cytochrome, ionic strength effects indicate a minus-minus interaction, with approximately a -2 charge near the site of reduction of the highest potential heme.     

Studies on the orientations of the mitochondrial redox carriers. II. Orientation of the mitochondrial chromophores with respect to the plane of the membrane in hydrated, oriented mitochondrial multilayers.

Orientations of the active site chromophores of the mitochondrial redox carriers have been investigated in hydrated, oriented multilayers of mitochondrial membranes using optical and EPR spectroscopy. The hemes of cytochrome c oxidase, cytochrome c1, and cytochromes b were found to be oriented in a similar manner, with the normal to their heme planes lying approximately in the plane of the mitochondrial membrane. The heme of cytochrome c was either less oriented in general or was oriented at an angle closer to the plane of the mitochondrial membrane than were the hemes of the "tightly bound" mitochondrial cytochromes. EPR spectra of the azide, sulfide and formate complexes of cytochrome c oxidase in mitochondria in situ obtained as a function of the orientation of the applied magnetic field relative to the planes of the membrane multilayers showed that both hemes of the oxidase were oriented in such a way that the angle between the heme normal and the membrane normal was approx. 90 degrees.     

Studies on the orientations of the mitochondrial redox carriers II. Orientation of the mitochondrial chromophores with respect to the plane of the membrane in hydrated,oriented mitochondrial multilayers

Orientations of the active site chromophores of the mitochondrial redox carriers have been investigated in hydrated, oriented multilayers of mitochondrial membranes using optical and EPR spectroscopy. The hemes of cytochrome c oxidase, cytochrome c₁, and cytochromes b were found to be oriented in a similar manner, with the normal to their heme planes lying approximately in the plane of the mitochondrial membrane. The heme of cytochrome c was either less oriented in general or was oriented at an angle closer to the plane of the mitochondrial membrane than were the hemes of the “tightly bound” mitochondrial cytochromes. EPR spectra of the azide, sulfide and formate complexes of cytochrome c oxidase in mitochondria in situ obtained as a function of the orientation of the applied magnetic field relative to the planes of the membrane multilayers showed that both hemes of the oxidase were oriented in such a way that the angle between the heme normal and the membrane normal was approx. 90°.     

Dielectric constant dependence of biological oxidation-reduction. 1. A model of polarity-dependent ferrocytochrome c oxidation

A theoretical model for the effect of the dielectric constant (c) of the solvent medium on ferrocytochrome c oxidation by ferricyanide is developed to account for the observed variations of the rate constant (k) of reactions in aqueous binary mixtures with alcohols (less than 5-10 mol% ethanol and propranolol). A correlation between k and c is found if ln k is expressed as a function of the Kirkwood parameter (c-1)(2c+1). The results of calculations indicate that the use of the 'overall dipole moment' of cytochrome c in oxidoreduction studies is likely to be unreliable. Instead, the decrease in k in alcohol/water mixtures is best explained--in conformity with Onsager's theory of the reaction field--by a polarity effect on the dipole moment of the cytochrome c heme upon diffusion of the polar solvent molecules into the low dielectric constant heme crevice.     

Antimicrobial activity and membrane selective interactions of a synthetic lipopeptide MSI-843

The method of fluorescence resonance energy transfer (FRET) has been employed to monitor cytochrome c interaction with bilayer phospholipid membranes. Liposomes composed of phosphatidylcholine and varying amounts of anionic lipid cardiolipin (CL) were used as model membranes. Trace amount of fluorescent lipid derivative, anthrylvinyl-phosphatidylcholine was incorporated into the membranes to serve energy donor for heme moiety of cytochrome c. Energy transfer efficiency was measured at different lipid and protein concentrations to obtain extensive set of data, which were further analyzed globally in terms of adequate models of protein adsorption and energy transfer on the membrane surface. It has been found that the cytochrome c association with membranes containing 10 mol% CL can be described in terms of equilibrium binding model (yielding dissociation constant Kd = 0.2-0.4 microM and stoichiometry n = 11-13 lipid molecules per protein binding site) combined with FRET model assuming uniform acceptor distribution with the distance of 3.5-3.6 nm between the bilayer midplane and heme moiety of cytochrome c. However, increasing the CL content to 20 or 40 mol% (at low ionic strength) resulted in a different behavior of FRET profiles, inconsistent with the concepts of equilibrium adsorption of cytochrome c at the membrane surface and/or uniform acceptor distribution. To explain this fact, several possibilities are analyzed, including cytochrome c-induced formation of non-bilayer structures and clusters of charged lipids, or changes in the depth of cytochrome c penetration into the bilayer depending on the protein surface density. Additional control experiments have shown that only the latter process can explain the peculiar concentration dependences of FRET at high CL content.     

Functional characterization of human duodenal cytochrome b (Cybrd1): Redox properties in relation to iron and ascorbate metabolism

Duodenal cytochrome b (Dcytb or Cybrd1) is an iron-regulated protein, highly expressed in the duodenal brush border membrane. It has ferric reductase activity and is believed to play a physiological role in dietary iron absorption. Its sequence identifies it as a member of the cytochrome b(561) family. A His-tagged construct of human Dcytb was expressed in insect Sf9 cells and purified. Yields of protein were increased by supplementation of the cells with 5-aminolevulinic acid to stimulate heme biosynthesis. Quantitative analysis of the recombinant Dcytb indicated two heme groups per monomer. Site-directed mutagenesis of any of the four conserved histidine residues (His 50, 86, 120 and 159) to alanine resulted in much diminished levels of heme in the purified Dcytb, while mutation of the non-conserved histidine 33 had no effect on the heme content. This indicates that those conserved histidines are heme ligands, and that the protein cannot stably bind heme if any of them is absent. Recombinant Dcytb was reduced by ascorbate under anaerobic conditions, the extent of reduction being 67% of that produced by dithionite. It was readily reoxidized by ferricyanide. EPR spectroscopy showed signals from low-spin ferriheme, consistent with bis-histidine coordination. These comprised a signal at gmax=3.7 corresponding to a highly anisotropic species, and another at gmax=3.18; these species are similar to those observed in other cytochromes of the b561 family, and were reducible by ascorbate. In addition another signal was observed in some preparations at gmax=2.95, but this was unreactive with ascorbate. Redox titrations indicated an average midpoint potential for the hemes in Dcytb of +80 mV+/-30 mV; the data are consistent with either two hemes at the same potential, or differing in potential by up to 60 mV. These results indicate that Dcytb is similar to the ascorbate-reducible cytochrome b561 of the adrenal chromaffin granule, though with some differences in midpoint potentials of the hemes.     

Membrane tetraheme cytochrome c(m552) of the ammonia-oxidizing nitrosomonas europaea: a ubiquinone reductase

Cytochrome c(m552) (cyt c(m552)) from the ammonia-oxidizing Nitrosomonas europaea is encoded by the cycB gene, which is preceded in a gene cluster by three genes encoding proteins involved in the oxidation of hydroxylamine: hao, hydroxylamine oxidoreductase; orf2, a putative membrane protein; cycA, cyt c(554). By amino acid sequence alignment of the core tetraheme domain, cyt c(m552) belongs to the NapC/TorC family of tetra- or pentaheme cytochrome c species involved in electron transport from membrane quinols to a variety of periplasmic electron shuttles leading to terminal reductases. However, cyt c(m552) is thought to reduce quinone with electrons originating from HAO. In this work, the tetrahemic 27 kDa cyt c(m552) from N. europaea was purified after extraction from membranes using Triton X-100 with subsequent exchange into n-dodecyl beta-d-maltoside. The cytochrome had a propensity to form strong SDS-resistant dimers likely mediated by a conserved GXXXG motif present in the putative transmembrane segment. Optical spectra of the ferric protein contained a broad ligand-metal charge transfer band at approximately 625 nm indicative of a high-spin heme. Mossbauer spectroscopy of the reduced (57)Fe-enriched protein revealed the presence of high-spin and low-spin hemes in a 1:3 ratio. Multimode EPR spectroscopy of the native state showed signals from an electronically interacting high-spin/low-spin pair of hemes. Upon partial reduction, a typical high-spin heme EPR signal was observed. No EPR signals were observed from the other two low-spin hemes, indicating an electronic interaction between these hemes as well. UV-vis absorption data indicate that CO (ferrous enzyme) or CN(-) (ferric or ferrous enzyme) bound to more than one and possibly all hemes. Other anionic ligands did not bind. The four ferrous hemes of the cytochrome were rapidly oxidized in the presence of oxygen. Comparative modeling, based on the crystal structure and conserved residues of the homologous NrfH protein from Desulfovibrio of cyt c(m552), predicted some structural elements, including a Met-ligated high-spin heme in a quinone-binding pocket, and likely axial ligands to all four hemes.     

Two hemes in Bacillus subtilis succinate:menaquinone oxidoreductase (complex II).

Succinate:menaquinone-7 oxidoreductase (complex II) of the Gram-positive bacterium Bacillus subtilis consists of equimolar amounts of three polypeptides; a 65-kDa FAD-containing polypeptide, a 28-kDa iron-sulfur cluster containing polypeptide, and a 23-kDa membrane-spanning cytochrome b558 polypeptide. The enzyme complex was overproduced 2-3-fold in membranes of B. subtilis cells containing the sdhCAB operon on a low copy number plasmid and was purified in the presence of detergent. The cytochrome b558 subunit alone was similarly overexpressed in a complex II deficient mutant and partially purified. Isolated complex II catalyzed the reduction of various quinones and also quinol oxidation. Both activities were efficiently albeit not completely blocked by 2-n-heptyl-4-hydroxyquinoline N-oxide. Chemical analysis demonstrated two protoheme IX per complex II. One heme component was found to have an Em,7.4 of +65 mV and an EPR gmax signal at 3.68, to be fully reducible by succinate, and showed a symmetrical alpha-band absorption peak at 555 nm at 77 K. The other heme component was found to have an Em,7.4 of -95 mV and an EPR gmax signal at 3.42, was not reducible by succinate under steady-state conditions, and showed in the reduced state an apparent split alpha-band absorption peak with maxima at 553 and 558 nm at 77 K. Potentiometric titrations of partially purified cytochrome b558 subunit demonstrated that the isolated cytochrome b558 also contains two hemes. Some of the properties, i.e., the alpha-band light absorption peak at 77 K, the line shapes of the EPR gmax signals, and reactivity with carbon monoxide were observed to be different in B. subtilis cytochrome b558 isolated and in complex II. This suggests that the bound flavoprotein and iron-sulfur protein subunits protect or affect the heme environment in the assembled complex.     

Spectral and potentiometric analysis of cytochromes from Bacillus subtilis

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