Catalytic Reactions and Energy Conservation in the Cytochrome bc1 and b6f Complexes of Energy-Transducing Membranes.

This review focuses on key components of respiratory and photosynthetic energy-transduction systems: the cytochrome bc1 and b6f (Cytbc1/b6f) membranous multisubunit homodimeric complexes. These remarkable molecular machines catalyze electron transfer from membranous quinones to water-soluble electron carriers (such as cytochromes c or plastocyanin), coupling electron flow to proton translocation across the energy-transducing membrane and contributing to the generation of a transmembrane electrochemical potential gradient, which powers cellular metabolism in the majority of living organisms. Cytsbc1/b6f share many similarities but also have significant differences. While decades of research have provided extensive knowledge on these enzymes, several important aspects of their molecular mechanisms remain to be elucidated. We summarize a broad range of structural, mechanistic, and physiological aspects required for function of Cytbc1/b6f, combining textbook fundamentals with new intriguing concepts that have emerged from more recent studies. The discussion covers but is not limited to (i) mechanisms of energy-conserving bifurcation of electron pathway and energy-wasting superoxide generation at the quinol oxidation site, (ii) the mechanism by which semiquinone is stabilized at the quinone reduction site, (iii) interactions with substrates and specific inhibitors, (iv) intermonomer electron transfer and the role of a dimeric complex, and (v) higher levels of organization and regulation that involve Cytsbc1/b6f. In addressing these topics, we point out existing uncertainties and controversies, which, as suggested, will drive further research in this field.

Isothermal titration calorimetry of membrane protein interactions: FNR and the cytochrome b6f complex.

Ferredoxin-NADP+ reductase (FNR) was previously inferred to bind to the cytochrome b6f complex in the electron transport chain of oxygenic photosynthesis. In the present study, this inference has been examined through analysis of the thermodynamics of the interaction between FNR and the b6f complex. Isothermal titration calorimetry (ITC) was used to characterize the physical interaction of FNR with b6f complex derived from two plant sources (Spinacia oleracea and Zea maize). ITC did not detect a significant interaction of FNR with the b6f complex in detergent solution nor with the complex reconstituted in liposomes. A previous inference of a small amplitude but defined FNR-b6f interaction is explained by FNR interaction with micelles of the undecyl ß-D maltoside (UDM) detergent micelles used to purify b6f. Circular dichroism, employed to analyze the effect of detergent on the FNR structure, did not reveal significant changes in secondary or tertiary structures of FNR domains in the presence of UDM detergent. However, thermodynamic analysis implied a significant decrease in an interaction between the N-terminal FAD-binding and C-terminal NADP+-binding domains of FNR caused by detergent. The enthalpy, ΔHo, and the entropy, ΔSo, associated with FNR unfolding decreased four-fold in the presence of 1 mM UDM at pH 6.5. In addition to the conclusion regarding the absence of a binding interaction of significant amplitude between FNR and the b6f complex, these studies provide a precedent for consideration of significant background protein-detergent interactions in ITC analyses involving integral membrane proteins.

Structural insight into the role of the Ton complex in energy transduction.

In Gram-negative bacteria, outer membrane transporters import nutrients by coupling to an inner membrane protein complex called the Ton complex. The Ton complex consists of TonB, ExbB, and ExbD, and uses the proton motive force at the inner membrane to transduce energy to the outer membrane via TonB. Here, we structurally characterize the Ton complex from Escherichia coli using X-ray crystallography, electron microscopy, double electron-electron resonance (DEER) spectroscopy, and crosslinking. Our results reveal a stoichiometry consisting of a pentamer of ExbB, a dimer of ExbD, and at least one TonB. Electrophysiology studies show that the Ton subcomplex forms pH-sensitive cation-selective channels and provide insight into the mechanism by which it may harness the proton motive force to produce energy.

Structural and functional contributions of lipids to the stability and activity of the photosynthetic cytochrome b6f lipoprotein complex.

The photosynthetic cytochrome b6f complex, a homodimer containing eight distinct subunits and 26 transmembrane helices per monomer, catalyzes proton-coupled electron transfer across the thylakoid membrane. The 2.5-Å-resolution structure of the complex from the cyanobacterium Nostoc sp. revealed the presence of 23 lipid-binding sites per monomer. Although the crystal structure of the cytochrome b6f from a plant source has not yet been solved, the identities of the lipids present in a plant b6f complex have previously been determined, indicating that the predominant lipid species are monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), phosphatidylglycerol (PG), and sulfoquinovosyldiacylglycerol (SQDG). Despite the extensive structural analyses of b6f-lipid interactions, the basis of the stabilization by lipids remains poorly understood. In the present study, we report on the effect of individual lipids on the structural and functional integrity of the b6f complex, purified from Spinacea oleracea It was found that (i) galactolipids (MGDG, DGDG, and SQDG) and phospholipids dilinolenoyl-phosphatidylglycerol (DLPG), 1,2-dioleoylphosphatidylglycerol (DOPG), and 1,2-dioleoyl-sn-glycerol-3-phosphatidylcholine (DOPC) structurally stabilize the complex to varying degrees; (ii) SQDG has a major role in stabilizing the dimeric complex; (iii) the b6f complex is stabilized by incorporation into nanodiscs or bicelles; (iv) removal of bound phospholipid by phospholipase A2 inactivates the cytochrome complex; and (v) activity can be restored significantly by the addition of the anionic lipid PG, which is attributed to stabilization of the quinone portal and the hinge region of the iron-sulfur protein.

Border crossings: colicins and transporters.

Colicins are protein toxins produced by Escherichia coli to kill related bacteria. They must cross the target cell outer membrane (OM), and some must also cross the inner membrane (IM). To accomplish cellular import, colicins have parasitized E. coli nutrient transporters as well as IM and periplasmic proteins normally used to maintain cell wall integrity or provide energy for nutrient uptake through transporters. Colicins have evolved to use both transporters and other membrane proteins through mechanisms different from those employed in physiological substrate uptake. Extended receptor-binding domains allow some colicins to search by lateral diffusion for binding sites on their OM translocators while bound to their primary OM receptor. Transport across the OM is initiated by entry of the unstructured N-terminal translocation domain into the translocator. Periplasmic and IM networks subsequently accomplish insertion of the colicin cytotoxic domain into or across the IM.

Metastable radical state, nonreactive with oxygen, is inherent to catalysis by respiratory and photosynthetic cytochromes bc1/b6f.

Oxygenic respiration and photosynthesis based on quinone redox reactions face a danger of wasteful energy dissipation by diversion of the productive electron transfer pathway through the generation of reactive oxygen species (ROS). Nevertheless, the widespread quinone oxido-reductases from the cytochrome bc family limit the amounts of released ROS to a low, perhaps just signaling, level through an as-yet-unknown mechanism. Here, we propose that a metastable radical state, nonreactive with oxygen, safely holds electrons at a local energetic minimum during the oxidation of plastohydroquinone catalyzed by the chloroplast cytochrome b6f This intermediate state is formed by interaction of a radical with a metal cofactor of a catalytic site. Modulation of its energy level on the energy landscape in photosynthetic vs. respiratory enzymes provides a possible mechanism to adjust electron transfer rates for efficient catalysis under different oxygen tensions.

Structure-function of the cytochrome b6f lipoprotein complex: a scientific odyssey and personal perspective.

Structure-function studies of the cytochrome b6f complex, the central hetero-oligomeric membrane protein complex in the electron transport chain of oxygenic photosynthesis, which formed the basis for a high-resolution (2.5 Å) crystallographic solution of the complex, are described. Structure-function differences between the structure of subunits of the bc complexes, b6f, and bc1 from mitochondria and photosynthetic bacteria, which are often assumed to function identically, are discussed. Major differences which suggest that quinone-dependent electron transport pathways can vary in b6f and bc1 complexes are as follows: (a) an additional c-type heme, cn, and bound single copies of chlorophyll a and ß-carotene in the b6f complex; and (b) a cyclic electron transport pathway that encompasses the b6f and PSI reaction center complexes. The importance of including lipid in crystallization of the cytochrome complex, or with any hetero-oligomeric membrane protein complex, is emphasized, and consequences to structure-function of b6f being a lipoprotein complex discussed, including intra-protein dielectric heterogeneity and resultant pathways of trans-membrane electron transport. The role of the b6f complex in trans-membrane signal transduction from reductant generated on the p-side of the electron transport chain to the regulation of light energy to the two photosystems by trans-side phosphorylation of the light-harvesting chlorophyll protein is presented. Regarding structure aspects relevant to plastoquinol-quinone entrance-egress: (i) modification of the p-side channel for plastoquinone access to the iron-sulfur protein would change the rate-limiting step in electron transport; (ii) the narrow niche for entry of plastoquinol into b6f from the PSII reaction center complex would seem to require close proximity between the complexes.

On mechanisms of colicin import: the outer membrane quandary.

Current problems in the understanding of colicin import across the Escherichia coli outer membrane (OM), involving a range of cytotoxic mechanisms, are discussed: (I) Crystal structure analysis of colicin E3 (RNAase) with bound OM vitamin B12 receptor, BtuB, and of the N-terminal translocation (T) domain of E3 and E9 (DNAase) inserted into the OM OmpF porin, provide details of the initial interaction of the colicin central receptor (R)- and N-terminal T-domain with OM receptors/translocators. (II) Features of the translocon include: (a) high-affinity (Kd ≈ 10-9 M) binding of the E3 receptor-binding R-domain E3 to BtuB; (b) insertion of disordered colicin N-terminal domain into the OmpF trimer; (c) binding of the N-terminus, documented for colicin E9, to the TolB protein on the periplasmic side of OmpF. Reinsertion of the colicin N-terminus into the second of the three pores in OmpF implies a colicin anchor site on the periplasmic side of OmpF. (III) Studies on the insertion of nuclease colicins into the cytoplasmic compartment imply that translocation proceeds via the C-terminal catalytic domain, proposed here to insert through the unoccupied third pore of the OmpF trimer, consistent with in vitro occlusion of OmpF channels by the isolated E3 C-terminal domain. (IV) Discussion of channel-forming colicins focuses mainly on colicin E1 for which BtuB is receptor and the OM TolC protein the proposed translocator. The ability of TolC, part of a multidrug efflux pump, for which there is no precedent for an import function, to provide a trans-periplasmic import pathway for colicin E1, is questioned on the basis of an unfavorable hairpin conformation of colicin N-terminal peptides inserted into TolC.

Trans-membrane Signaling in Photosynthetic State Transitions: REDOX- AND STRUCTURE-DEPENDENT INTERACTION IN VITRO BETWEEN STT7 KINASE AND THE CYTOCHROME b6f COMPLEX.

Trans-membrane signaling involving a serine/threonine kinase (Stt7 in Chlamydomonas reinhardtii) directs light energy distribution between the two photosystems of oxygenic photosynthesis. Oxidation of plastoquinol mediated by the cytochrome b6f complex on the electrochemically positive side of the thylakoid membrane activates the kinase domain of Stt7 on the trans (negative) side, leading to phosphorylation and redistribution ("state transition") of the light-harvesting chlorophyll proteins between the two photosystems. The molecular description of the Stt7 kinase and its interaction with the cytochrome b6f complex are unknown or unclear. In this study, Stt7 kinase has been cloned, expressed, and purified in a heterologous host. Stt7 kinase is shown to be active in vitro in the presence of reductant and purified as a tetramer, as determined by analytical ultracentrifugation, electron microscopy, and electrospray ionization mass spectrometry, with a molecular weight of 332 kDa, consisting of an 83.41-kDa monomer. Far-UV circular dichroism spectra show Stt7 to be mostly α-helical and document a physical interaction with the b6f complex through increased thermal stability of Stt7 secondary structure. The activity of wild-type Stt7 and its Cys-Ser mutant at positions 68 and 73 in the presence of a reductant suggest that the enzyme does not require a disulfide bridge for its activity as suggested elsewhere. Kinase activation in vivo could result from direct interaction between Stt7 and the b6f complex or long-range reduction of Stt7 by superoxide, known to be generated in the b6f complex by quinol oxidation.

David W. Krogmann, 1931-2016.

We provide here reflections on the life and career of David W. Krogmann (1931-2016), a great scientist, a mentor and an outstanding teacher, who had a remarkable impact on anyone who came in contact with him. Dave was a pillar of photosynthesis at Purdue University, and an international authority on electron transfer intermediates in oxygenic photosynthesis, particularly the soluble cytochromes. The photosynthetic system of his choice was cyanobacteria, and one of his major discoveries was the Orange Carotenoid Protein in these microrganisms.

The Colicin E1 TolC-Binding Conformer: Pillar or Pore Function of TolC in Colicin Import?

The mechanism by which the drug export protein TolC is utilized for import of the cytotoxin colicin E1 across the outer membrane and periplasmic space is addressed. Studies of the initial binding of colicin E1 with TolC, occlusion of membrane-incorporated TolC ion channels, and the structure underlying the colicin-TolC complex were based on the interactions with TolC of individual colicin translocation domain (T-domain) peptides from a set of 19 that span different segments of the T-domain. These studies led to identification of a short 20-residue segment 101-120, a "TolC box", located near the center of the colicin T-domain, which is necessary for binding of colicin to TolC. Omission of this segment eliminated the ability of the T-domain to occlude TolC channels and to co-elute with TolC on a size-exclusion column. Far-ultraviolet circular dichroism spectral and thermal stability analysis of the structure of T-domain peptides implies (i) a helical hairpin conformation of the T-domain, (ii) the overlap of the TolC-binding site with a hinge of the helical hairpin, and (iii) a TolC-dependent stage of colicin import in which a central segment of the T-domain in a helical hairpin conformation binds to the TolC entry port following initial binding to the BtuB receptor. These studies provide the first structure-based information about the interaction of colicin E1 with the unique TolC protein. The model inferred for binding of the T-domain to TolC implies reservations about the traditional model for colicin import in which TolC functions to provide a channel for translocation of the colicin in an unfolded state across the bacterial outer membrane and a large part of the periplasmic space.

Photo-induced oxidation of the uniquely liganded heme f in the cytochrome b6f complex of oxygenic photosynthesis.

The ultrafast behavior of the ferrous heme f from the cytochrome b6f complex of oxygenic photosynthesis is revealed by means of transient absorption spectroscopy. Benefiting from the use of microfluidic technologies for handling the sample as well as from a complementary frame-by-frame analysis of the heme dynamics, the different relaxation mechanisms from vibrationally excited states are disentangled and monitored via the shifts of the heme α-absorption band. Under 520 nm laser excitation, about 85% of the heme f undergoes pulse-limited photo-oxidation (<100 fs), with the electron acceptor being most probably one of the adjacent aromatic amino acid residues. After charge recombination in 5.3 ps, the residual excess energy is dissipated in 3.6 ps. In a parallel pathway, the remaining 15% of the hemes directly relax from their excited state in 2.5 ps. In contrast to a vast variety of heme-proteins, including the homologous heme c1 from the cytochrome bc1 complex, there is no evidence that heme f photo-dissociates from its axial ligands. Due to its unique binding, with histidine and an unusual tyrosine as axial ligands, the heme f exemplifies a dependence of ultrafast dynamics on the structural environment.

Quinone-dependent proton transfer pathways in the photosynthetic cytochrome b6f complex.

As much as two-thirds of the proton gradient used for transmembrane free energy storage in oxygenic photosynthesis is generated by the cytochrome b6f complex. The proton uptake pathway from the electrochemically negative (n) aqueous phase to the n-side quinone binding site of the complex, and a probable route for proton exit to the positive phase resulting from quinol oxidation, are defined in a 2.70-Å crystal structure and in structures with quinone analog inhibitors at 3.07 Å (tridecyl-stigmatellin) and 3.25-Å (2-nonyl-4-hydroxyquinoline N-oxide) resolution. The simplest n-side proton pathway extends from the aqueous phase via Asp20 and Arg207 (cytochrome b6 subunit) to quinone bound axially to heme c(n). On the positive side, the heme-proximal Glu78 (subunit IV), which accepts protons from plastosemiquinone, defines a route for H(+) transfer to the aqueous phase. These pathways provide a structure-based description of the quinone-mediated proton transfer responsible for generation of the transmembrane electrochemical potential gradient in oxygenic photosynthesis.

Role of domain swapping in the hetero-oligomeric cytochrome b6f lipoprotein complex.

Domain swapping that contributes to the stability of biologically crucial multisubunit complexes has been implicated in protein oligomerization. In the case of membrane protein assemblies, domain swapping of the iron-sulfur protein (ISP) subunit occurs in the hetero-oligomeric cytochrome b6f and bc1 complexes, which are organized as symmetric dimers that generate the transmembrane proton electrochemical gradient utilized for ATP synthesis. In these complexes, the ISP C-terminal predominantly ß-sheet extrinsic domain containing the redox-active [2Fe-2S] cluster resides on the electrochemically positive side of each monomer in the dimeric complex. This domain is bound to the membrane sector of the complex through an N-terminal transmembrane α-helix that is "swapped' to the other monomer of the complex where it spans the complex and the membrane. Detailed analysis of the function and structure of the b6f complex isolated from the cyanobacterium Fremyella diplosiphon SF33 shows that the domain-swapped ISP structure is necessary for function but is not necessarily essential for maintenance of the dimeric structure of the complex. On the basis of crystal structures of the cytochrome complex, the stability of the cytochrome dimer is attributed to specific intermonomer protein-protein and protein-lipid hydrophobic interactions. The geometry of the domain-swapped ISP structure is proposed to be a consequence of the requirement that the anchoring helix of the ISP not perturb the heme organization or quinone channel in the conserved core of each monomer.

Traffic within the cytochrome b6f lipoprotein complex: gating of the quinone portal.

The cytochrome bc complexes b6f and bc1 catalyze proton-coupled quinol/quinone redox reactions to generate a transmembrane proton electrochemical gradient. Quinol oxidation on the electrochemically positive (p) interface of the complex occurs at the end of a narrow quinol/quinone entry/exit Qp portal, 11 Å long in bc complexes. Superoxide, which has multiple signaling functions, is a by-product of the p-side quinol oxidation. Although the transmembrane core and the chemistry of quinone redox reactions are conserved in bc complexes, the rate of superoxide generation is an order of magnitude greater in the b6f complex, implying that functionally significant differences in structure exist between the b6f and bc1 complexes on the p-side. A unique structure feature of the b6f p-side quinol oxidation site is the presence of a single chlorophyll-a molecule whose function is unrelated to light harvesting. This study describes a cocrystal structure of the cytochrome b6f complex with the quinol analog stigmatellin, which partitions in the Qp portal of the bc1 complex, but not effectively in b6f. It is inferred that the Qp portal is partially occluded in the b6f complex relative to bc1. Based on a discrete molecular-dynamics analysis, occlusion of the Qp portal is attributed to the presence of the chlorophyll phytyl tail, which increases the quinone residence time within the Qp portal and is inferred to be a cause of enhanced superoxide production. This study attributes a novel (to our knowledge), structure-linked function to the otherwise enigmatic chlorophyll-a in the b6f complex, which may also be relevant to intracellular redox signaling.

Transmembrane signaling and assembly of the cytochrome b6f-lipidic charge transfer complex.

Structure-function properties of the cytochrome b6f complex are sufficiently unique compared to those of the cytochrome bc1 complex that b6f should not be considered a trivially modified bc1 complex. A unique property of the dimeric b6f complex is its involvement in transmembrane signaling associated with the p-side oxidation of plastoquinol. Structure analysis of lipid binding sites in the cyanobacterial b6f complex prepared by hydrophobic chromatography shows that the space occupied by the H transmembrane helix in the cytochrome b subunit of the bc1 complex is mostly filled by a lipid in the b6f crystal structure. It is suggested that this space can be filled by the domain of a transmembrane signaling protein. The identification of lipid sites and likely function defines the intra-membrane conserved central core of the b6f complex, consisting of the seven trans-membrane helices of the cytochrome b and subunit IV polypeptides. The other six TM helices, contributed by cytochrome f, the iron-sulfur protein, and the four peripheral single span subunits, define a peripheral less conserved domain of the complex. The distribution of conserved and non-conserved domains of each monomer of the complex, and the position and inferred function of a number of the lipids, suggests a model for the sequential assembly in the membrane of the eight subunits of the b6f complex, in which the assembly is initiated by formation of the cytochrome b6-subunit IV core sub-complex in a monomer unit. Two conformations of the unique lipidic chlorophyll a, defined in crystal structures, are described, and functions of the outlying ß-carotene, a possible 'latch' in supercomplex formation, are discussed. This article is part of a Special Issue entitled: Respiratory complex III and related bc complexes.

Mechanism of enhanced superoxide production in the cytochrome b(6)f complex of oxygenic photosynthesis.

The specific rate of superoxide (O2(•-)) production in the purified active crystallizable cytochrome b6f complex, normalized to the rate of electron transport, has been found to be more than an order of magnitude greater than that measured in isolated yeast respiratory bc1 complex. The biochemical and structural basis for the enhanced production of O2(•-) in the cytochrome b6f complex compared to that in the bc1 complex is discussed. The higher rate of superoxide production in the b6f complex could be a consequence of an increased residence time of plastosemiquinone/plastoquinol in its binding niche near the Rieske protein iron-sulfur cluster, resulting from (i) occlusion of the quinone portal by the phytyl chain of the unique bound chlorophyll, (ii) an altered environment of the proton-accepting glutamate believed to be a proton acceptor from semiquinone, or (iii) a more negative redox potential of the heme bp on the electrochemically positive side of the complex. The enhanced rate of superoxide production in the b6f complex is physiologically significant as the chloroplast-generated reactive oxygen species (ROS) functions in the regulation of excess excitation energy, is a source of oxidative damage inflicted during photosynthetic reactions, and is a major source of ROS in plant cells. Altered levels of ROS production are believed to convey redox signaling from the organelle to the cytosol and nucleus.

Lipid-induced conformational changes within the cytochrome b6f complex of oxygenic photosynthesis.

Cytochrome b6f catalyzes quinone redox reactions within photosynthetic membranes to generate a transmembrane proton electrochemical gradient for ATP synthesis. A key step involves the transfer of an electron from the [2Fe-2S] cluster of the iron-sulfur protein (ISP) extrinsic domain to the cytochrome f heme across a distance of 26 Å, which is too large for competent electron transfer but could be bridged by translation-rotation of the ISP. Here we report the first crystallographic evidence of significant motion of the ISP extrinsic domain. It is inferred that extensive crystallographic disorder of the ISP extrinsic domain indicates conformational flexibility. The ISP disorder observed in this structure, in contrast to the largely ordered ISP structure observed in the b6f complex supplemented with neutral lipids, is attributed to electrostatic interactions arising from anionic lipids.

The Q cycle of cytochrome bc complexes: a structure perspective.

Aspects of the crystal structures of the hetero-oligomeric cytochrome bc(1) and b(6)f ("bc") complexes relevant to their electron/proton transfer function and the associated redox reactions of the lipophilic quinones are discussed. Differences between the b(6)f and bc(1) complexes are emphasized. The cytochrome bc(1) and b(6)f dimeric complexes diverge in structure from a core of subunits that coordinate redox groups consisting of two bis-histidine coordinated hemes, a heme b(n) and b(p) on the electrochemically negative (n) and positive (p) sides of the complex, the high potential [2Fe-2S] cluster and c-type heme at the p-side aqueous interface and aqueous phase, respectively, and quinone/quinol binding sites on the n- and p-sides of the complex. The bc(1) and b(6)f complexes diverge in subunit composition and structure away from this core. b(6)f Also contains additional prosthetic groups including a c-type heme c(n) on the n-side, and a chlorophyll a and ß-carotene. Common structure aspects; functions of the symmetric dimer. (I) Quinone exchange with the bilayer. An inter-monomer protein-free cavity of approximately 30Å along the membrane normal×25Å (central inter-monomer distance)×15Å (depth in the center), is common to both bc(1) and b(6)f complexes, providing a niche in which the lipophilic quinone/quinol (Q/QH(2)) can be exchanged with the membrane bilayer. (II) Electron transfer. The dimeric structure and the proximity of the two hemes b(p) on the electrochemically positive side of the complex in the two monomer units allow the possibility of two alternate routes of electron transfer across the complex from heme b(p) to b(n): intra-monomer and inter-monomer involving electron cross-over between the two hemes b(p). A structure-based summary of inter-heme distances in seven bc complexes, representing mitochondrial, chromatophore, cyanobacterial, and algal sources, indicates that, based on the distance parameter, the intra-monomer pathway would be favored kinetically. (III) Separation of quinone binding sites. A consequence of the dimer structure and the position of the Q/QH(2) binding sites is that the p-side QH(2) oxidation and n-side Q reduction sites are each well separated. Therefore, in the event of an overlap in residence time by QH(2) or Q molecules at the two oxidation or reduction sites, their spatial separation would result in minimal steric interference between extended Q or QH(2) isoprenoid chains. (IV) Trans-membrane QH(2)/Q transfer. (i) n/p-side QH(2)/Q transfer may be hindered by lipid acyl chains; (ii) the shorter less hindered inter-monomer pathway across the complex would not pass through the center of the cavity, as inferred from the n-side antimycin site on one monomer and the p-side stigmatellin site on the other residing on the same surface of the complex. (V) Narrow p-side portal for QH(2)/Q passage. The [2Fe-2S] cluster that serves as oxidant, and whose histidine ligand serves as a H(+) acceptor in the oxidation of QH(2), is connected to the inter-monomer cavity by a narrow extended portal, which is also occupied in the b(6)f complex by the 20 carbon phytyl chain of the bound chlorophyll.

Crystal structures of the OmpF porin: function in a colicin translocon.

The OmpF porin in the Escherichia coli outer membrane (OM) is required for the cytotoxic action of group A colicins, which are proposed to insert their translocation and active domains through OmpF pores. A crystal structure was sought of OmpF with an inserted colicin segment. A 1.6 A OmpF structure, obtained from crystals formed in 1 M Mg2+, has one Mg2+ bound in the selectivity filter between Asp113 and Glu117 of loop 3. Co-crystallization of OmpF with the unfolded 83 residue glycine-rich N-terminal segment of colicin E3 (T83) that occludes OmpF ion channels yielded a 3.0 A structure with inserted T83, which was obtained without Mg2+ as was T83 binding to OmpF. The incremental electron density could be modelled as an extended poly-glycine peptide of at least seven residues. It overlapped the Mg2+ binding site obtained without T83, explaining the absence of peptide binding in the presence of Mg2+. Involvement of OmpF in colicin passage through the OM was further documented by immuno-extraction of an OM complex, the colicin translocon, consisting of colicin E3, BtuB and OmpF.