The YedZ family: possible heme binding proteins that can be fused to transporters and electron carriers

YedZ of Escherichia coli is an integral 6 transmembrane spanning (TMS) protein of unknown function. We have identified homologues of YedZ in bacteria and animals but could not find homologues in Archaea or the other eukaryotic kingdoms. YedZ homologues exhibit conserved histidyl residues in their transmembrane domains that may function in heme binding. Some of the homologues encoded in the genomes of magnetotactic bacteria and cyanobacteria have YedZ domains fused to transport and electron transfer proteins, respectively. One of the animal homologues is the 6 TMS epithelial plasma membrane antigen of the prostate (STAMP1) that is overexpressed in prostate cancer. Animal homologues have YedZ domains fused C-terminal to homologues of coenzyme F420-dependent NADP oxidoreductases. YedZ homologues are shown to have arisen by intragenic triplication of a 2 TMS-encoding element. They exhibit slight but statistically significant sequence similarity to two families of putative heme export systems and one family of cytochrome-containing electron carriers. We propose that YedZ homologues function as heme-binding proteins that can facilitate or regulate oxidoreduction, transmembrane electron flow and transport.     

Synthesis and characterization of de novo designed peptides modelling the binding sites of [4Fe-4S] clusters in photosystem I

Photosystem I (PS I) converts the energy of light into chemical energy via transmembrane charge separation. The terminal electron transfer cofactors in PS I are three low-potential [4Fe-4S] clusters named F_X, F_A and F_B, the last two are bound by the PsaC subunit. We have modelled the F_A and F_B binding sites by preparing two apo-peptides (maquettes), sixteen amino acids each. These model peptides incorporate the consensus [4Fe-4S] binding motif along with amino acids from the immediate environment of the iron-sulfur clusters F_A and F_B. The [4Fe-4S] clusters were successfully incorporated into these model peptides, as shown by optical absorbance, EPR and Mössbauer spectroscopies. The oxidation-reduction potential of the iron-sulfur cluster in the F_A-maquette is − 0.44 ± 0.03 V and in the F_B-maquette is − 0.47 ± 0.03 V. Both are close to that of F_A and F_B in PS I and are considerably more negative than that observed for other [4Fe-4S] model systems described earlier (Gibney, B. R., Mulholland, S. E., Rabanal, F., and Dutton, P. L. Proc. Natl. Acad. Sci. U.S.A. 93 (1996) 15041-15046). Our optical data show that both maquettes can irreversibly bind to PS I complexes, where PsaC-bound F_A and F_B were removed, and possibly participate in the light-induced electron transfer reaction in PS I.     

Ero1-α and PDIs constitute a hierarchical electron transfer network of endoplasmic reticulum oxidoreductases

Ero1-α and endoplasmic reticulum (ER) oxidoreductases of the protein disulfide isomerase (PDI) family promote the efficient introduction of disulfide bonds into nascent polypeptides in the ER. However, the hierarchy of electron transfer among these oxidoreductases is poorly understood. In this paper, Ero1-α–associated oxidoreductases were identified by proteomic analysis and further confirmed by surface plasmon resonance. Ero1-α and PDI were found to constitute a regulatory hub, whereby PDI induced conformational flexibility in an Ero1-α shuttle cysteine (Cys99) facilitated intramolecular electron transfer to the active site. In isolation, Ero1-α also oxidized ERp46, ERp57, and P5; however, kinetic measurements and redox equilibrium analysis revealed that PDI preferentially oxidized other oxidoreductases. PDI accepted electrons from the other oxidoreductases via its a′ domain, bypassing the a domain, which serves as the electron acceptor from reduced glutathione. These observations provide an integrated picture of the hierarchy of cooperative redox interactions among ER oxidoreductases in mammalian cells.     

Histidine cycle mechanism for the concerted proton/electron transfer from ascorbate to the cytosolic haem b centre of cytochrome b561: a unique machinery for the biological transmembrane electron transfer

Cytochromes b(561) are a family of transmembrane proteins found in most eukaryotic cells and contain two haem b prosthetic groups per molecule being coordinated with four His residues from four different transmembrane alpha-helices. Although cytochromes b(561) residing in the chromaffin vesicles has long been known to have a role for a neuroendocrine-specific transmembrane electron transfer from extravesicular ascorbate to intravesicular monodehydroascorbate radical to regenerate ascorbate, newly found members were apparently lacking in the sequence for putative ascorbate-binding site but exhibiting a transmembrane ferrireductase activity. We propose that cytochrome b(561) has a specific mechanism to facilitate the concerted proton/electron transfer from ascorbate by exploiting a cycle of deprotonated and protonated states of the N(delta1) atom of the axial His residue at the extravesicular haem center, as an initial step of the transmembrane electron transfer. This mechanism utilizes the well-known electrochemistry of ascorbate for a biological transmembrane electron transfer and might be operative for other type of electron transfer reactions from organic reductants.     

Design of amphiphilic protein maquettes: enhancing maquette functionality through binding of extremely hydrophobic cofactors to lipophilic domains

We demonstrate coordination of the extremely hydrophobic 13(2)-OH-Ni-bacteriochlorophyll (Ni-BChl) to the lipophilic domain of a novel, designed amphiphilic protein maquette (AP3) dispersed in detergent micelles [Discher et al. (2005) Biochemistry 44, 12329-12343]. Sedimentation velocity and equilibrium experiments and steady-state absorption spectra indicate that Ni-BChl-AP3 is a four-helix bundle containing one Ni-BChl axially ligated by one or two histidines. The nature of the ligation was pursued with ultrafast visible spectroscopy. While it is well established that light excitation of axially ligated mono- and bisimidazole Ni-BChl in solution leads to rapid imidazole dissociation and nanosecond recombination, there is no evidence of axial ligand dissociation in the light-excited Ni-BChl-AP3. This indicates that Ni-BChl is confined within the AP3 protein, ligated to histidines with severely restricted mobility. Dissociation constants show that Ni-BChl binding to AP3 is considerably weaker than the nanomolar range usual for heme and hydrophilic (HP) maquettes; moreover, there is a tendency for the Ni-BChl-AP3 four-helix bundles to dimerize into eight-helix bundles. Nevertheless, the preparation of the Ni-BChl-AP3 four-alpha-helix maquettes, supported by time-resolved spectroscopic analysis of the nature of the ligation, provides a viable new approach to AP maquette designs that address the challenges involved in binding extremely hydrophobic cofactors.     

Wolinella succinogenes quinol:fumarate reductase-2.2-A resolution crystal structure and the E-pathway hypothesis of coupled transmembrane proton and electron transfer

The structure of the respiratory membrane protein complex quinol:fumarate reductase (QFR) from Wolinella succinogenes has been determined by X-ray crystallography at 2.2-A resolution [Nature 402 (1999) 377]. Based on the structure of the three protein subunits A, B, and C and the arrangement of the six prosthetic groups (a covalently bound FAD, three iron-sulfur clusters, and two haem b groups), a pathway of electron transfer from the quinol-oxidising dihaem cytochrome b in the membrane to the site of fumarate reduction in the hydrophilic subunit A has been proposed. The structure of the membrane-integral dihaem cytochrome b reveals that all transmembrane helical segments are tilted with respect to the membrane normal. The "four-helix" dihaem binding motif is very different from other dihaem-binding transmembrane four-helix bundles, such as the "two-helix motif" of the cytochrome bc(1) complex and the "three-helix motif" of the formate dehydrogenase/hydrogenase group. The gamma-hydroxyl group of Ser C141 has an important role in stabilising a kink in transmembrane helix IV. By combining the results from site-directed mutagenesis, functional and electrochemical characterisation, and X-ray crystallography, a residue was identified which was found to be essential for menaquinol oxidation [Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 13051]. The distal location of this residue in the structure indicates that the coupling of the oxidation of menaquinol to the reduction of fumarate in dihaem-containing succinate:quinone oxidoreductases could in principle be associated with the generation of a transmembrane electrochemical potential. However, it is suggested here that in W. succinogenes QFR, this electrogenic effect is counterbalanced by the transfer of two protons via a proton transfer pathway (the "E-pathway") in concert with the transfer of two electrons via the membrane-bound haem groups. According to this "E-pathway hypothesis", the net reaction catalysed by W. succinogenes QFR does not contribute directly to the generation of a transmembrane electrochemical potential.     

Evidence for transmembrane proton transfer in a dihaem-containing membrane protein complex

Membrane protein complexes can support both the generation and utilisation of a transmembrane electrochemical proton potential ('proton-motive force'), either by transmembrane electron transfer coupled to protolytic reactions on opposite sides of the membrane or by transmembrane proton transfer. Here we provide the first evidence that both of these mechanisms are combined in the case of a specific respiratory membrane protein complex, the dihaem-containing quinol:fumarate reductase (QFR) of Wolinella succinogenes, so as to facilitate transmembrane electron transfer by transmembrane proton transfer. We also demonstrate the non-functionality of this novel transmembrane proton transfer pathway ('E-pathway') in a variant QFR where a key glutamate residue has been replaced. The 'E-pathway', discussed on the basis of the 1.78-Angstrom-resolution crystal structure of QFR, can be concluded to be essential also for the viability of pathogenic varepsilon-proteobacteria such as Helicobacter pylori and is possibly relevant to proton transfer in other dihaem-containing membrane proteins, performing very different physiological functions.     

Enhanced translocation of amphiphilic peptides across membranes by transmembrane proteins

Cell membranes are phospholipid bilayers with a large number of embedded transmembrane proteins. Some of these proteins, such as scramblases, have properties that facilitate lipid flip-flop from one membrane leaflet to another. Scramblases and similar transmembrane proteins could also affect the translocation of other amphiphilic molecules, including cell-penetrating or antimicrobial peptides. We studied the effect of transmembrane proteins on the translocation of amphiphilic peptides through the membrane. Using two very different models, we consistently demonstrate that transmembrane proteins with a hydrophilic patch enhance the translocation of amphiphilic peptides by stabilizing the peptide in the membrane. Moreover, there is an optimum amphiphilicity because the peptide could become overstabilized in the transmembrane state, in which the peptide-protein dissociation is hampered, limiting the peptide translocation. The presence of scramblases and other proteins with similar properties could be exploited for more efficient transport into cells. The described principles could also be utilized in the design of a drug-delivery system by the addition of a translocation-enhancing peptide that would integrate into the membrane.     

Succinate:quinone oxidoreductases--what can we learn from Wolinella succinogenes quinol:fumarate reductase?

The structure of Wolinella succinogenes quinol:fumarate reductase by X-ray crystallography has been determined at 2.2-A resolution [Lancaster et al. (1999), Nature 402, 377-385]. Based on the structure of the three protein subunits A, B, and C and the arrangement of the six prosthetic groups (a covalently bound FAD, three iron-sulphur clusters, and two haem b groups) a pathway of electron transfer from the quinol-oxidising dihaem cytochrome b in the membrane to the site of fumarate reduction in the hydrophilic subunit A has been proposed. By combining the results from site-directed mutagenesis, functional and electrochemical characterisation, and X-ray crystallography, a residue was identified which is essential for menaquinol oxidation. [Lancaster et al. (2000), Proc. Natl. Acad. Sci. USA 97, 13051-13056]. The location of this residue in the structure suggests that the coupling of the oxidation of menaquinol to the reduction of fumarate in dihaem-containing succinate:quinone oxidoreductases could be associated with the generation of a transmembrane electrochemical potential. Based on crystallographic analysis of three different crystal forms of the enzyme and the results from site-directed mutagenesis, we have derived a mechanism of fumarate reduction and succinate oxidation [Lancaster et al. (2001) Eur. J. Biochem. 268, 1820-1827], which should be generally relevant throughout the superfamily of succinate:quinone oxidoreductases.     

Semidehydroascorbic acid as an intermediate in norepinephrine biosynthesis in chromaffin granules.

We investigated whether semidehydroascorbic acid was an intermediate in norepinephrine synthesis in chromaffin granules and in electron transfer across the chromaffin granule membrane. Semidehydroascorbic acid was measured in intact granules by electron spin resonance. In the presence of intragranular but not extragranular ascorbic acid, semidehydroascorbic acid was formed within granules in direct relationship to dopamine beta-monooxygenase activity. However, semidehydroascorbic acid was not generated when granules were incubated with epinephrine instead of the substrate dopamine, with dopamine beta-monooxygenase inhibitors, without oxygen, and when intragranular ascorbic acid was depleted. Experiments using the impermeant paramagnetic broadening agents [K3 [Cr(C2O4)3].3H2O] and Ni(en)3(NO3)2 provided further evidence that semidehydroascorbic acid was generated only within granules. We also investigated semidehydroascorbic acid formation in the presence of intragranular and extragranular ascorbic acid. Under these conditions, semidehydroascorbic acid was formed on both sides of the granule membrane, and formation was coupled to dopamine beta-monooxygenase activity. These data indicate that dopamine beta-monooxygenase is reduced by single electron transfer from intragranular ascorbic acid, that transmembrane electron transfer occurs by single electron transfer, and that transmembrane electron transfer is directly coupled to formation of intragranular semidehydroascorbic acid via dopamine beta-monooxygenase activity.     

Structure,functionality and tuning up of laccases for lignocellulose and other industrial applications

Laccases (EC 1.10.3.2) are copper-containing oxidoreductases that have a relatively high redox potential which enables them to catalyze oxidation of phenolic compounds, including lignin-derived phenolics. The laccase-catalyzed oxidation of phenolics is accompanied by concomitant reduction of dioxygen to water via copper catalysis and involves a series of electron transfer reactions balanced by a stepwise re-oxidation of copper ions in the active site of the enzyme. The reaction details of the catalytic four-copper mechanism of laccase-mediated catalysis are carefully re-examined and clarified. The substrate range for laccase catalysis can be expanded by means of supplementary mediators that essentially function as vehicles for electron transfer. Comparisons of amino acid sequences and structural traits of selected laccases reveal conservation of the active site trinuclear center geometry but differences in loop conformations. We also evaluate the features and regions of laccases in relation to modification and evolution of laccases for various industrial applications including lignocellulosic biomass processing.     

Wolinella succinogenes quinol:fumarate reductase—2.2-Å resolution crystal structure and the E-pathway hypothesis of coupled transmembrane proton and electron transfer

The structure of the respiratory membrane protein complex quinol:fumarate reductase (QFR) from Wolinella succinogenes has been determined by X-ray crystallography at 2.2-Å resolution [Nature 402 (1999) 377]. Based on the structure of the three protein subunits A, B, and C and the arrangement of the six prosthetic groups (a covalently bound FAD, three iron-sulfur clusters, and two haem b groups), a pathway of electron transfer from the quinol-oxidising dihaem cytochrome b in the membrane to the site of fumarate reduction in the hydrophilic subunit A has been proposed. The structure of the membrane-integral dihaem cytochrome b reveals that all transmembrane helical segments are tilted with respect to the membrane normal. The “four-helix” dihaem binding motif is very different from other dihaem-binding transmembrane four-helix bundles, such as the “two-helix motif” of the cytochrome bc₁ complex and the “three-helix motif” of the formate dehydrogenase/hydrogenase group. The γ-hydroxyl group of Ser C141 has an important role in stabilising a kink in transmembrane helix IV. By combining the results from site-directed mutagenesis, functional and electrochemical characterisation, and X-ray crystallography, a residue was identified which was found to be essential for menaquinol oxidation [Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 13051]. The distal location of this residue in the structure indicates that the coupling of the oxidation of menaquinol to the reduction of fumarate in dihaem-containing succinate:quinone oxidoreductases could in principle be associated with the generation of a transmembrane electrochemical potential. However, it is suggested here that in W. succinogenes QFR, this electrogenic effect is counterbalanced by the transfer of two protons via a proton transfer pathway (the “E-pathway”) in concert with the transfer of two electrons via the membrane-bound haem groups. According to this “E-pathway hypothesis”, the net reaction catalysed by W. succinogenes QFR does not contribute directly to the generation of a transmembrane electrochemical potential.     

Glucose enzyme electrode using cytochrome b(562) as an electron mediator

We demonstrate the construction of glucose sensors employing pyrroloquinoline quinone (PQQ) glucose dehydrogenase (PQQGDH) from Acinetobacter calcoaceticus and glucose oxidase (GOD) from Aspergillus nigar coupled with Escherichia coli soluble cytochrome b(562) (cyt b(562)) as electron acceptor. PQQGDH and GOD do not show direct electrochemical recycling of the prosthetic group at the electrode surface leading to a corresponding current signal. We constructed PQQGDH and GOD electrodes co-immobilized with 100-fold molar excess of cyt b(562) and investigated the electrochemical properties without synthetic electron mediators. PQQGDH/cyt b(562) and GOD/cyt b(562) electrodes both responded well to glucose whereas no current increase was observed from the electrode immobilizing enzyme alone. The detection limits for the PQQGDH/cyt b(562) and GOD/cyt b(562) electrodes were 0.1 and 0.8 mM, respectively, and their linearity extended to over 2 and 9 mM, respectively. These results demonstrate that a sensor system can be constructed without a synthetic electron mediator by using a natural electron acceptor. Furthermore, we have demonstrated the potential application of cyt b(562) in direct electron transfer type sensor systems with oxidoreductases whose quaternary structure do not contain any electron transfer subunit.     

Diversity and origin of alternative NADH:ubiquinone oxidoreductases

Mitochondria from various organisms, especially plants, fungi and many bacteria contain so-called alternative NADH:ubiquinone oxidoreductases that catalyse the same redox reaction as respiratory chain complex I, but do not contribute to the generation of transmembrane proton gradients. In eucaryotes, these enzymes are associated with the mitochondrial inner membrane, with their NADH reaction site facing either the mitochondrial matrix (internal alternative NADH:ubiquinone oxidoreductases) or the cytoplasm (external alternative NADH:ubiquinone oxidoreductases). Some of these enzymes also accept NADPH as substrate, some require calcium for activity. In the past few years, the characterisation of several alternative NADH:ubiquinone oxidoreductases on the DNA and on the protein level, of substrate specificities, mitochondrial import and targeting to the mitochondrial inner membrane has greatly improved our understanding of these enzymes. The present review will, with an emphasis on yeast model systems, illuminate various aspects of the biochemistry of alternative NADH:ubiquinone oxidoreductases, address recent developments and discuss some of the questions still open in the field.     

Purified cytochrome b561 catalyzes transmembrane electron transfer for dopamine beta-hydroxylase and peptidyl glycine alpha-amidating monooxygenase activities in reconstituted systems

Cytochrome b561 from bovine adrenal medulla chromaffin granules has been purified by fast protein liquid chromatography chromatofocusing. The purified cytochrome was reconstituted into ascorbate-loaded phosphatidylcholine vesicles. With this reconstituted system transmembrane electron transfer for extravesicular soluble dopamine beta-hydroxylase activity was demonstrated. In accordance with the model proposed by Njus et al. (Njus, D., Knoth, J., Cook, C., and Kelley, P. M. (1983) J. Biol. Chem. 258, 27-30), catalytic amounts of a redox mediator were necessary to achieve electron transfer between cytochrome and soluble dopamine beta-hydroxylase. Our observations also showed that when membranous dopamine beta-hydroxylase was reconstituted on cytochrome containing vesicles, electron transfer occurred only in the presence of a redox mediator. Since cytochrome b561 has been found in secretory vesicles associated with peptidyl glycine alpha-amidating monooxygenase, electron transfer to this enzyme was also examined. Analogous to the results obtained for dopamine beta-hydroxylase, transmembrane electron transfer to peptidyl glycine alpha-amidating monooxygenase appears to require a redox mediator between cytochrome and this monooxygenase. These observations indicate that purified cytochrome b561 is capable of providing a transmembrane supply of electrons for both monooxygenases. Since no direct protein to protein electron transfer occurs, the results support the hypothesis that the ascorbate/semidehydroascorbate redox pair serves as a mediator for these enzymes in vivo.     

Zinc inhibition of electron transfer: mechanism of beta receptor inhibition?

Zinc has been shown to inhibit β-receptor activation of adenylate cyclase at a post receptor site. We have postulated that the β-receptor is one of several receptors activated by reduction, followed by transmembrane elector transfer accelerated by GTP. GTP accelerates electron transfer in a model system and this accelerated electron transfer is inhibited by zinc. This could explain the mechanism of the post receptor inhibition by zinc of the adenylate cyclase stimulation which follows β-receptor activation.     

Radical reactions of thiamin pyrophosphate in 2-oxoacid oxidoreductases

Thiamin pyrophosphate (TPP) is essential in carbohydrate metabolism in all forms of life. TPP-dependent decarboxylation reactions of 2-oxo-acid substrates result in enamine adducts between the thiazolium moiety of the coenzyme and decarboxylated substrate. These central enamine intermediates experience different fates from protonation in pyruvate decarboxylase to oxidation by the 2-oxoacid dehydrogenase complexes, the pyruvate oxidases, and 2-oxoacid oxidoreductases. Virtually all of the TPP-dependent enzymes, including pyruvate decarboxylase, can be assayed by 1-electron redox reactions linked to ferricyanide. Oxidation of the enamines is thought to occur via a 2-electron process in the 2-oxoacid dehydrogenase complexes, wherein acyl group transfer is associated with reduction of the disulfide of the lipoamide moiety. However, discrete 1-electron steps occur in the oxidoreductases, where one or more [4Fe-4S] clusters mediate the electron transfer reactions to external electron acceptors. These radical intermediates can be detected in the absence of the acyl-group acceptor, coenzyme A (CoASH). The π-electron system of the thiazolium ring stabilizes the radical. The extensively delocalized character of the radical is evidenced by quantitative analysis of nuclear hyperfine splitting tensors as detected by electron paramagnetic resonance (EPR) spectroscopy and by electronic structure calculations. The second electron transfer step is markedly accelerated by the presence of CoASH. While details of the second electron transfer step and its facilitation by CoASH remain elusive, expected redox properties of potential intermediates limit possible scenarios. This article is part of a Special Issue entitled: Radical SAM enzymes and Radical Enzymology.