Conservation of lipid functions in cytochrome bc complexes

Lipid binding sites and properties are compared in two sub-families of hetero-oligomeric membrane protein complexes known to have similar functions in order to gain further understanding of the role of lipid in the function, dynamics, and assembly of these complexes. Using the crystal structure information for both complexes, we compared the lipid binding properties of the cytochrome b₆f and bc₁ complexes that function in photosynthetic and respiratory membrane energy transduction. Comparison of lipid and detergent binding sites in the b₆f complex with those in bc₁ shows significant conservation of lipid positions. Seven lipid binding sites in the cyanobacterial b₆f complex overlap three natural sites in the Chlamydomonas reinhardtii algal complex and four sites in the yeast mitochondrial bc₁ complex. The specific identity of lipids is different in b₆f and bc₁ complexes: b₆f contains sulfoquinovosyldiacylglycerol, phosphatidylglycerol, phosphatidylcholine, monogalactosyldiacylglycerol, and digalactosyldiacylglycerol, whereas cardiolipin, phosphatidylethanolamine, and phosphatidic acid are present in the yeast bc₁ complex. The lipidic chlorophyll a and β-carotene (β-car) in cyanobacterial b₆f, as well as eicosane in C. reinhardtii, are unique to the b₆f complex. Inferences of lipid binding sites and functions were supported by sequence, interatomic distance, and B-factor information on interacting lipid groups and coordinating amino acid residues. The lipid functions inferred in the b₆f complex are as follows: (i) substitution of a transmembrane helix by a lipid and chlorin ring, (ii) lipid and β-car connection of peripheral and core domains, (iii) stabilization of the iron-sulfur protein transmembrane helix, (iv) n-side charge and polarity compensation, and (v) β-car-mediated super-complex with the photosystem I complex.     

Formation of engineered intersubunit disulfide bond in cytochrome bc1 complex disrupts electron transfer activity in the complex

Protein domain movement of the Rieske iron-sulfur protein has been speculated to play an essential role in the bifurcated oxidation of ubiquinol catalyzed by the cytochrome bc₁ complex. To better understand the electron transfer mechanism of the bifurcated ubiquinol oxidation at Qp site, we fixed the head domain of ISP at the cyt c₁ position by creating an intersubunit disulfide bond between two genetically engineered cysteine residues: one at position 141 of ISP and the other at position 180 of the cyt c₁ [S141C(ISP)/G180C(cyt c₁)]. The formation of a disulfide bond between ISP and cyt c₁ in this mutant complex is confirmed by SDS-PAGE and Western blot. In this mutant complex, the disulfide bond formation is concurrent with the loss of the electron transfer activity of the complex. When the disulfide bond is released by treatment with β-mercaptoethanol, the activity is restored. These results further support the hypothesis that the mobility of the head domain of ISP is functionally important in the cytochrome bc₁ complex. Formation of the disulfide bond between ISP and cyt c₁ shortens the distance between the [2Fe-2S] cluster and heme c₁, hence the rate of intersubunit electron transfer between these two redox prosthetic groups induced by pH change is increased. The intersubunit disulfide bond formation also decreases the rate of stigmatellin induced reduction of ISP in the fully oxidized complex, suggesting that an endogenous electron donor comes from the vicinity of the b position in the cytochrome b.     

Domain conformational switch of the iron–sulfur protein in cytochrome bc1 complex is induced by the electron transfer from cytochrome bL to bH

Intensive biochemical, biophysical and structural studies of the cytochrome (cyt) bc₁ complex in the past have led to the formulation of the “protonmotive Q-cycle” mechanism for electron and proton transfer in this vitally important complex. The key step of this mechanism is the separation of electrons during the oxidation of a substrate quinol at the Q_P site with both electrons transferred simultaneously to ISP and cyt b_L when the extrinsic domain of ISP (ISP-ED) is located at the b-position. Pre-steady state fast kinetic analysis of bc₁ demonstrates that the reduced ISP-ED moves to the c₁-position to reduce cyt c₁ only after the reduced cyt b_L is oxidized by cyt b_H. However, the question of how the conformational switch of ISP-ED is initiated remains unanswered. The results obtained from analysis of inhibitory efficacy and binding affinity of two types of Q_P site inhibitors, Pm and Pf, under various redox states of the bc₁ complex, suggest that the electron transfer from heme b_L to b_H is the driving force for the releasing of the reduced ISP-ED from the b-position to c₁-position to reduce cyt c₁.     

The cytochrome b lysine 329 residue is critical for ubihydroquinone oxidation and proton release at the Qo site of bacterial cytochrome bc1

The ubihydroquinone:cytochrome (cyt) c oxidoreductase (or cyt bc₁) is an important enzyme for photosynthesis and respiration. In bacteria like Rhodobacter capsulatus, this membrane complex has three subunits, the iron?sulfur protein (ISP) with its Fe₂S₂ cluster, cyt c₁ and cyt b, forming two catalytic domains, the Q_o (hydroquinone (QH₂) oxidation) and Q_i (quinone (Q) reduction) sites. At the Q_o site, the electron transfer pathways originating from QH₂ oxidation are known, but their associated proton release routes are less well defined. Earlier, we demonstrated that the His291 of cyt b is important for this latter process. In this work, using the bacterial cyt bc₁ and site directed mutagenesis, we show that Lys329 of cyt b is also critical for electron and proton transfer at the Q_o site. Of the mutants examined, Lys329Arg was photosynthesis proficient and had quasi-wild type cyt bc₁ activity. In contrast, the Lys329Ala and Lys329Asp were photosynthesis-impaired and contained defective but assembled cyt bc₁. In particular, the bifurcated electron transfer and associated proton(s) release reactions occurring during QH₂ oxidation were drastically impaired in Lys329Asp mutant. Furthermore, in silico docking studies showed that in this mutant the location and the H-bonding network around the Fe₂S₂ cluster of ISP on cyt b surface was different than the wild type enzyme. Based on these experimental findings and theoretical considerations, we propose that the presence of a positive charge at position 329 of cyt b is critical for efficient electron transfer and proton release for QH₂ oxidation at the Q_o site of cyt bc₁.     

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 Q_p 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 Q_p portal of the bc₁ complex, but not effectively in b₆f. It is inferred that the Q_p portal is partially occluded in the b₆f complex relative to bc₁. Based on a discrete molecular-dynamics analysis, occlusion of the Q_p portal is attributed to the presence of the chlorophyll phytyl tail, which increases the quinone residence time within the Q_p 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.     

Studies on Inhibition of Respiratory Cytochrome bc 1 Complex by the Fungicide Pyrimorph Suggest a Novel Inhibitory Mechanism

The respiratory chain cytochrome bc ₁ complex (cyt bc ₁) is a major target of numerous antibiotics and fungicides. All cyt bc ₁ inhibitors act on either the ubiquinol oxidation (Q_P) or ubiquinone reduction (Q_N) site. The primary cause of resistance to bc ₁ inhibitors is target site mutations, creating a need for novel agents that act on alternative sites within the cyt bc ₁ to overcome resistance. Pyrimorph, a synthetic fungicide, inhibits the growth of a broad range of plant pathogenic fungi, though little is known concerning its mechanism of action. In this study, using isolated mitochondria from pathogenic fungus Phytophthora capsici, we show that pyrimorph blocks mitochondrial electron transport by affecting the function of cyt bc ₁. Indeed, pyrimorph inhibits the activities of both purified 11-subunit mitochondrial and 4-subunit bacterial bc ₁ with IC₅₀ values of 85.0 μM and 69.2 μM, respectively, indicating that it targets the essential subunits of cyt bc ₁ complexes. Using an array of biochemical and spectral methods, we show that pyrimorph acts on an area near the Q_P site and falls into the category of a mixed-type, noncompetitive inhibitor with respect to the substrate ubiquinol. In silico molecular docking of pyrimorph to cyt b from mammalian and bacterial sources also suggests that pyrimorph binds in the vicinity of the quinol oxidation site.     

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 Q_p 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 Q_p portal of the bc₁ complex, but not effectively in b₆f. It is inferred that the Q_p portal is partially occluded in the b₆f complex relative to bc₁. Based on a discrete molecular-dynamics analysis, occlusion of the Q_p portal is attributed to the presence of the chlorophyll phytyl tail, which increases the quinone residence time within the Q_p 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.     

Comparison of the Cytochrome bc 1 Complex with theAnticipated Structure of the Cytochrome b 6 f Complex:De Plus Ça Change de Plus C'est la Même Chose

Structural alignment of the integral cytochrome b ₆-SU IV subunits with the solved structure of themitochondrial bc ₁ complex shows a pronounced asymmetry. There is a much higher homology onthe p-side of the membrane, suggesting a similarity in the mechanisms of intramembrane andinterfacial electron and proton transfer on the p-side, but not necessarily on the n-side. Structuraldifferences between the bc ₁ and b ₆ f complexes appear to be larger the farther the domain or subunitis removed from the membrane core, with extreme differences between cytochromes c ₁ and f. Aspecial role for the dimer may involve electron sharing between the two hemes b _p, which is indicatedas a probable event by calculations of relative rate constants for intramonomer heme b _p hemeb _n, or intermonomer heme b _p heme b _p electron transfer. The long-standing observation offlash-induced oxidation of only 0.5 of the chemical content of cyt f may be partly a consequence ofthe statistical population of ISP bound to cyt f on the dimer. It is proposed that the p-side domainof cyt f is positioned with its long axis parallel to the membrane surface in order to: (i) allow itslarge and small domains to carry out the functions of cyt c ₁ and suVIII, respectively, of the bc ₁complex, and (ii) provide maximum dielectric continuity with the membrane. (iii) This positionwould also allow the internal water chain (proton wire) of cyt f to serve as the p-side exit portfor an intramembrane H⁺ transfer chain that would deprotonate the semiquinol located in themyxothiazol/MOA-stilbene pocket near heme b _p. A hypothesis is presented for the identity of theamino acid residues in this chain.     

Probing the local lipid environment of the Rhodobacter sphaeroides cytochrome bc1 and Synechocystis sp. PCC 6803 cytochrome b6f complexes with styrene maleic acid

Intracytoplasmic vesicles (chromatophores) in the photosynthetic bacterium Rhodobacter sphaeroides represent a minimal structural and functional unit for absorbing photons and utilising their energy for the generation of ATP. The cytochrome bc₁ complex (cytbc₁) is one of the four major components of the chromatophore alongside the reaction centre-light harvesting 1-PufX core complex (RC-LH1-PufX), the light-harvesting 2 complex (LH2), and ATP synthase. Although the membrane organisation of these complexes is known, their local lipid environments have not been investigated. Here we utilise poly(styrene-alt-maleic acid) (SMA) co-polymers as a tool to simultaneously determine the local lipid environments of the RC-LH1-PufX, LH2 and cytbc₁ complexes. SMA has previously been reported to effectively solubilise complexes in lipid-rich membrane regions whilst leaving lipid-poor ordered protein arrays intact. Here we show that SMA solubilises cytbc₁ complexes with an efficiency of nearly 70%, whereas solubilisation of RC-LH1-PufX and LH2 was only 10% and 22% respectively. This high susceptibility of cytbc₁ to SMA solubilisation is consistent with this complex residing in a locally lipid-rich region. SMA solubilised cytbc₁ complexes retain their native dimeric structure and co-purify with 56 ± 6 phospholipids from the chromatophore membrane. We extended this approach to the model cyanobacterium Synechocystis sp. PCC 6803, and show that the cytochrome b₆f complex (cytb₆f) and Photosystem II (PSII) complexes are susceptible to SMA solubilisation, suggesting they also reside in lipid-rich environments. Thus, lipid-rich membrane regions could be a general requirement for cytbc₁/cytb₆f complexes, providing a favourable local solvent to promote rapid quinol/quinone binding and release at the Q₀ and Q_i sites.     

Chlorophyll a is the crucial redox sensor and transmembrane signal transmitter in the cytochrome b6f complex. Components and mechanisms of state transitions from the hydrophobic mismatch viewpoint

The cytochrome (cyt) b₆f complex is involved in the transmembrane redox signaling that triggers state transitions in cyanobacteria and chloroplasts. However, the components and molecular mechanisms are still unclear. In an attempt to solve this long-standing problem, we first focused on the unknown role of a single chlorophyll a (Chla) in cyt b₆f with a new approach based on Chla structural properties. Various b₆f X-ray crystal structures were analyzed to identify their differences, which correlate with differences in Chla molecular volume. We found that the distance of the Rieske [2Fe-2S] cluster to Chla correlates with the distance between a pair of residues at the Q_o-site and the distance between a pair of residues at the opposite membrane side. These correlations were accompanied by the rotation of a key peripheral residue and by changes in the hydrophobic thickness of cyt b₆f. Parallel analysis of cyt bc₁ crystal structures allowed us to conclude that Chla acts as the crucial redox sensor and transmembrane signal transmitter in b₆f for changes in the plastoquinone pool redox state. The hydrophobic mismatch induced by the changed hydrophobic thickness of cyt b₆f is the driving force for the structural reorganizations of the photosynthetic apparatus during induction and the progression of state transitions in cyanobacteria and chloroplasts. A mechanism for LHCII kinase activation in chloroplasts is also proposed. Our understanding of the dynamic structural changes in bc-complexes during turnover at the Q_o-site and state transitions is augmented by the time-sequence ordering of 56 bc crystal structures.     

Spectral analysis of the bc1 complex components in situ: Beyond the traditional difference approach

The cytochrome (cyt) bc₁ complex (ubiquinol: cytochrome c oxidoreductase) is the central enzyme of mitochondrial and bacterial electron-transport chains. It is rich in prosthetic groups, many of which have significant but overlapping absorption bands in the visible spectrum. The kinetics of the cytochrome components of the bc₁ complex are traditionally followed by using the difference of absorbance changes at two or more different wavelengths. This difference-wavelength (DW) approach has been used extensively in the development and testing of the Q-cycle mechanism of the bc₁ complex in Rhodobacter sphaeroides chromatophores. However, the DW approach does not fully compensate for spectral interference from other components, which can significantly distort both amplitudes and kinetics. Mechanistic elaboration of cyt bc₁ turnover requires an approach that overcomes this limitation. Here, we compare the traditional DW approach to a least squares (LS) analysis of electron transport, based on newly determined difference spectra of all individual components of cyclic electron transport in chromatophores. Multiple sets of kinetic traces, measured at different wavelengths in the absence and presence of specific inhibitors, were analyzed by both LS and DW approaches. Comparison of the two methods showed that the DW approach did not adequately correct for the spectral overlap among the components, and was generally unreliable when amplitude changes for a component of interest were small. In particular, it was unable to correct for extraneous contributions to the amplitudes and kinetics of cyt b_L. From LS analysis of the chromophoric components (RC, c_tot, b_H and b_L), we show that while the Q-cycle model remains firmly grounded, quantitative reevaluation of rates, amplitudes, delays, etc., of individual components is necessary. We conclude that further exploration of mechanisms of the bc₁ complex, will require LS deconvolution for reliable measurement of the kinetics of individual components of the complex in situ.     

Quinone Pathways in Entire Photosynthetic Chromatophores of Rhodospirillum photometricum

In photosynthetic organisms, membrane pigment-protein complexes [light-harvesting complex 1 (LH1) and light-harvesting complex 2 (LH2)] harvest solar energy and convert sunlight into an electrical and redox potential gradient (reaction center) with high efficiency. Recent atomic force microscopy studies have described their organization in native membranes. However, the cytochrome (cyt) bc₁ complex remains unseen, and the important question of how reduction energy can efficiently pass from core complexes (reaction center and LH1) to distant cyt bc₁ via membrane-soluble quinones needs to be addressed. Here, we report atomic force microscopy images of entire chromatophores of Rhodospirillum photometricum. We found that core complexes influence their molecular environment within a critical radius of ∼ 250 Å. Due to the size mismatch with LH2, lipid membrane spaces favorable for quinone diffusion are found within this critical radius around cores. We show that core complexes form a network throughout entire chromatophores, providing potential quinone diffusion pathways that will considerably speed the redox energy transfer to distant cyt bc₁. These long-range quinone pathway networks result from cooperative short-range interactions of cores with their immediate environment.     

Spectral and kinetic resolution of the bc1 complex components in situ: A simple and robust alternative to the traditional difference wavelength approach

The kinetics of the cytochrome (cyt) components of the bc₁ complex (ubiquinol: cytochrome c oxidoreductase, Complex III) are traditionally followed by using the difference of absorbance changes at two or more different wavelengths. However, this difference-wavelength (DW) approach is of limited accuracy in the separation of absorbance changes of components with overlapping spectral bands. To resolve the kinetics of individual components in Rhodobacter sphaeroides chromatophores, we have tested a simplified version of a least squares (LS) analysis, based on measurement at a minimal number of different wavelengths. The success of the simplified LS analysis depended significantly on the wavelengths used in the set. The “traditional” set of 6 wavelengths (542, 551, 561, 566, 569 and 575 nm), normally used in the DW approach to characterize kinetics of cyt c_tot (cyt c₁ + cyt c₂), cyt b_L, cyt b_H, and P870 in chromatophores, could also be used to determine these components via the simplified LS analysis, with improved resolution of the individual components. However, this set is not sufficient when information about cyts c₁ and c₂ is needed. We identified multiple alternative sets of 5 and 6 wavelengths that could be used to determine the kinetics of all 5 components (P870 and cyts c₁, c₂, b_L, and b_H) simultaneously, with an accuracy comparable to that of the LS analysis based on a full set of wavelengths (1 nm intervals). We conclude that a simplified version of LS deconvolution based on a small number of carefully selected wavelengths provides a robust and significant improvement over the traditional DW approach, since it accounts for spectral interference of the different components, and uses fewer measurements when information about all five individual components is needed. Using the simplified and complete LS analyses, we measured the simultaneous kinetics of all cytochrome components of bc₁ complex in the absence and presence of specific inhibitors and found that they correspond well to those expected from the modified Q-cycle. This is the first study in which the kinetics of all cytochrome and reaction center components of the bc₁ complex functioning in situ have been measured simultaneously, with full deconvolution over an extended time range.     

Structural Basis of Multifunctional Bovine Mitochondrial Cytochrome bc 1 Complex

The mitochondrial cytochrome bc ₁ complex is a multifunctional membrane protein complex. Itcatalyzes electron transfer, proton translocation, peptide processing, and superoxide generation.Crystal structure data at 2.9 Å resolution not only establishes the location of the redox centersand inhibitor binding sites, but also suggests a movement of the head domain of the iron–sulfurprotein (ISP) during bc ₁ catalysis and inhibition of peptide-processing activity during complexmaturation. The functional importance of the movement of extramembrane (head) domain ofISP in the bc ₁ complex is confirmed by analysis of the Rhodobacter sphaeroides bc ₁ complexmutants with increased rigidity in the ISP neck and by the determination of rate constants foracid/base-induced intramolecular electron transfer between [2Fe–2S] and heme c ₁ in nativeand inhibitor-loaded beef complexes. The peptide-processing activity is activated in bovineheart mitochondrial bc ₁ complex by nonionic detergent at concentrations that inactivate electrontransfer activity. This peptide-processing activity is shown to be associated with subunits Iand II by cloning, overexpression and in vitro reconstitution. The superoxide-generation siteof the cytochrome bc ₁ complex is located at reduced b _L and Q^–. The reaction is membranepotential-, and cytochrome c-dependent.     

The role of the membrane bound cytochromes of b- and c-type in the electron transport chain of Rhodobacter capsulatus

(1) The electron transport system of heterotrophically dark-grown Rhodobacter capsulatus was investigated using the wild-type strain MT1131 and the phototrophic non-competent (Ps^-) mutant MT-GS18 carrying deletions of the genes for cytochrome c ₁ and b of the bc ₁ complex and for cytochrome c ₂. (2) Spectroscopic and thermodynamic data demonstrate that deletion of both bc ₁ complex and cyt. c ₂ still leaves several haems of c- and b-type with Em_7.0 of +265 mV and +354 mV at 551–542 nm, and +415 mV and +275 mV at 561–575 nm, respectively. (3) Analysis of the oxidoreduction kinetic patterns of cytochromes indicated that cyt. b ₄₁₅ and cyt. b ₂₇₅ are reduced by either ascorbate-diaminodurene or NADH, respectively. (4) Growth on different carbon and nitrogen sources revealed that the membrane-bound electron transport chain of both MT1131 and MT-GS18 strains undergoes functional modifications in response to the composition of the growth medium used. (5) Excitation of membrane fragments from cells grown in malate minimal medium by a train of single turnover flashes of light led to a rapid oxidation of 32% of the membrane-bound c-type haem complement. Conversely, membranes prepared from peptone/yeast extract grown cells did not show cyt. c photooxidation. These results are discussed within the framework of an electron transport chain in which alternative pathways bypassing both the cyt. c ₂ and bc ₁ complex might involve high-potential membrane bound haems of b- and c-type.Abbreviations AA antimycin A - CCCP carbonylcyanide m-chlorophenyl hydrazone - CN^- cyanide - DAD diaminodurene - Q₂H₂ ubiquinol-2 - Q-pool ubiquinone-10 pool - RC photochemical reaction center     

Photoinduced electron transfer in cytochrome bc1: Dynamics of rotation of the Iron-sulfur protein during bifurcated electron transfer from ubiquinol to cytochrome c1 and cytochrome bL

The electron transfer reactions within wild-type Rhodobacter sphaeroides cytochrome bc₁ (cyt bc₁) were studied using a binuclear ruthenium complex to rapidly photooxidize cyt c₁. When cyt c₁, the iron?sulfur center Fe₂S₂, and cyt b_H were reduced before the reaction, photooxidation of cyt c₁ led to electron transfer from Fe₂S₂ to cyt c₁ with a rate constant of k_a = 80,000 s^?1, followed by bifurcated reduction of both Fe₂S₂ and cyt b_L by QH₂ in the Q_o site with a rate constant of k₂ = 3000 s^?1. The resulting Q then traveled from the Q_o site to the Q_i site and oxidized one equivalent each of cyt b_L and cyt b_H with a rate constant of k₃ = 340 s^?1. The rate constant k_a was decreased in a nonlinear fashion by a factor of 53 as the viscosity was increased to 13.7. A mechanism that is consistent with the effect of viscosity involves rotational diffusion of the iron?sulfur protein from the b state with reduced Fe₂S₂ close to cyt b_L to one or more intermediate states, followed by rotation to the final c₁ state with Fe₂S₂ close to cyt c₁, and rapid electron transfer to cyt c₁.     

The α-helical membrane spanning domain of cytochrome b5 interacts with cytochrome P450 via nonspecific interactions

Cytochrome b₅ (cyt b₅) is an amphipathic membrane-bound heme protein found in the endoplasmic reticulum of eukaryotes. It consists of three domains, an N-terminal cytosolic, hydrophilic domain containing the heme, a short flexible linker and an α-helical membrane-spanning domain. This study investigated whether there are specific side chain helix–helix packing interactions between the COOH-terminal membrane anchor of cyt b₅ and cytochrome P450 (cyt P450) 2B4 in a purified reconstituted system. Alanine was inserted at six positions in the membrane anchor of cyt b₅. Insertion of alanine into an α-helix causes all amino acids at its carboxyl terminus to be rotated by 100°. The ability of the alanine insertion mutants of cyt b₅ to bind to cyt P450 2B4 was similar to that of the wild-type protein as was the ability of the mutant cyts b₅ to stimulate the metabolism of the anesthetic, methoxyflurane. These results demonstrate that the C-terminal hydrophobic α-helix of cyt b₅ does not interact with cyt P450 2B4 through a specific stereochemical fit of amino acid side chains, but rather through nonspecific interactions.     

Quantification of energy-converting protein complexes in plant thylakoid membranes

Knowledge about the exact abundance and ratio of photosynthetic protein complexes in thylakoid membranes is central to understanding structure-function relationships in energy conversion. Recent modeling approaches for studying light harvesting and electron transport reactions rely on quantitative information on the constituent complexes in thylakoid membranes. Over the last decades several quantitative methods have been established and refined, enabling precise stoichiometric information on the five main energy-converting building blocks in the thylakoid membrane: Light-harvesting complex II (LHCII), Photosystem II (PSII), Photosystem I (PSI), cytochrome b₆f complex (cyt b₆f complex), and ATPase. This paper summarizes a few quantitative spectroscopic and biochemical methods that are currently available for quantification of plant thylakoid protein complexes. Two new methods are presented for quantification of LHCII and the cyt b₆f complex, which agree well with established methods. In addition, recent improvements in mass spectrometry (MS) allow deeper compositional information on thylakoid membranes. The comparison between mass spectrometric and more classical protein quantification methods shows similar quantities of complexes, confirming the potential of thylakoid protein complex quantification by MS. The quantitative information on PSII, PSI, and LHCII reveal that about one third of LHCII must be associated with PSI for a balanced light energy absorption by the two photosystems.     

Environmental Challenges and Physiological Solutions: Comparative Energetic Daily Rhythms of Field Mice Populations from Different Ecosystems

Daily and seasonal variations in physiological characteristics of mammals can be considered adaptations to temporal habitat variables. Across different ecosystems, physiological adjustments are expected to be sensitive to different environmental signals such as changes in photoperiod, temperature or water and food availability; the relative importance of a particular signal being dependent on the ecosystem in question. Energy intake, oxygen consumption (VO₂) and body temperature (T_b) daily rhythms were compared between two populations of the broad-toothed field mouse Apodemus mystacinus, one from a Mediterranean and another from a sub-Alpine ecosystem. Mice were acclimated to short-day (SD) ‘winter’ and long-day (LD) ‘summer’ photoperiods under different levels of salinity simulating osmotic challenges. Mediterranean mice had higher VO₂ values than sub-Alpine mice. In addition, mice exposed to short days had higher VO₂ values when given water with a high salinity compared with mice exposed to long days. By comparison, across both populations, increasing salinity resulted in a decreased T_b in SD- but not in LD-mice. Thus, SD-mice may conserve energy by decreasing T_b during (‘winter’) conditions which are expected to be cool, whereas LD-mice might do the opposite and maintain a higher T_b during (‘summer’) conditions which are expected to be warm. LD-mice behaved to reduce energy expenditure, which might be considered a useful trait during ‘summer’ conditions. Overall, increasing salinity was a clear signal for Mediterranean-mice with resultant effects on VO₂ and T_b daily rhythms but had less of an effect on sub-Alpine mice, which were more responsive to changes in photoperiod. Results provide an insight into how different populations respond physiologically to various environmental challenges.     

Localization of photosynthetic electron transport components in mesophyll and bundle sheath chloroplasts of Zea mays

The organization of the electron transport components in mesophyll and bundle sheath chloroplasts of Zea mays was investigated. Grana-containing mesophyll chloroplasts (chlorophyll a to chlorophyll b ratio of about 3.0) possessed the full complement of the various electron transport components, comparable to chloroplasts from C₃ plants. Agranal bundle sheath chloroplasts ($\text{Chl}\text{a}\text{Chl}\text{b}\text{> 5.0}$) contained the full complement of photosystem (PS) I and of cytochrome (cyt) f but lacked a major portion of PS II and its associated Chl $\text{a}\text{b}$ light-harvesting complex (LHC), and most of the cyt b₅₅₉. The kinetic analysis of system I photoactivity revealed that the functional photosynthetic unit size of PS I was unchanged and identical in mesophyll and bundle sheath chloroplasts. The results suggest that PS I is contained in stroma-exposed thylakoids and that it does not receive excitation energy from the Chl $\text{a}\text{b}$ LHC present in the grana. A stoichiometric parity between PS I and cyt f in mesophyll and bundle sheath chloroplasts indicates that biosynthetic and functional properties of cyt f and P₇₀₀ are closely coordinated. Thus, it is likely that both cyt f and P₇₀₀ are located in the membrane of the intergrana thylakoids only. The kinetic analysis of PS II photoactivity revealed the absence of PS II_αfrom the bundle sheath chloroplasts and helped identify the small complement of system II in bundle sheath chloroplasts as PS II_β. The distribution of the main electron transport components in grana and stroma thylakoids is presented in a model of the higher plant chloroplast membrane system.