Lateral mobility of cytochrome c on intact mitochondrial membranes as determined by fluorescence redistribution after photobleaching.

Lateral mobility of an active fluorescent derivative of cytochrome c on the membranes of giant mitochondria was measured by fluorescence redistribution after photobleaching. A diffusion coefficient of 1.6 X 10(-10) cm2/sec was determined for the labeled cytochrome c on inner mitochondrial membranes under conditions where succinate oxidase activity was demonstrated. This relatively low rate of diffusion, together with results of other investigators, is explained in terms of a model involving a dynamic equilibrium between freely diffusing and associated forms of electron-transfer components.     

Spectral and redox characterization of the heme ci of the cytochrome b6f complex

Absorption spectra of the purified cytochrome b(6)f complex from Chlamydomonas reinhardtii were monitored as a function of the redox potential. Four spectral and redox components were identified: in addition to heme f and the two b hemes, the fourth component must be the new heme c(i) (also denoted x) recently discovered in the crystallographic structures. This heme is covalently attached to the protein, but has no amino acid axial ligand. It is located in the plastoquinone-reducing site Q(i) in the immediate vicinity of a b heme. Each heme titrated as a one-electron Nernst curve, with midpoint potentials at pH 7.0 of -130 mV and -35 mV (hemes b), +100 mV (heme c(i)), and +355 mV (heme f). The reduced minus oxidized spectrum of heme c(i) consists of a broad absorption increase centered approximately 425 nm. Its potential has a dependence of -60 mV/pH unit, implying that the reduced form binds one proton in the pH 6-9 range. The Q(i) site inhibitor 2-n-nonyl-4-hydroxyquinoline N-oxide, a semiquinone analogue, induces a shift of this potential by about -225 mV. The spectrum of c(i) matches the absorption changes previously observed in vivo for an unknown redox center denoted "G." The data are discussed with respect to the effect of the membrane potential on the electron transfer equilibrium between G and heme b(H) found in earlier experiments.     

Apparent dependence of interactions between cytochrome b5 and cytochrome b5 reductase upon translational diffusion in dimyristoyl lecithin liposomes.

Dimyristoyl lecithin liposomes, containing cytochrome b5 reductase (NADH:ferricytochrome b5 oxidoreductase, EC 1.6.2.2) and varying amounts of cytochrome b5, were used to measure flavoprotein catalysis alone and catalysis requiring electron transfer between the reductase and cytochrome as a function of temperature. Whereas flavoprotein catalysis showed a simple linear temperature dependence in an Arrhenius plot, the reaction involving electron transfer between the two bound enzymes showed a marked, 4-fold, change in rate at the crystalline-liquid crystalline phase transition of the hydrocarbon chains of the lecithin vesicles and a second, minor change involving the minor transition. These data represent strong evidence that protein-protein interactions in this membrane model system are dependent upon translational diffusion of nonpolar segments of the proteins in the hydrocarbon region of the phospholipid bilayer.     

Intraprotein transfer of the quinone analogue inhibitor 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone in the cytochrome b6f complex

Details are presented of the structural analysis of the cytochrome b(6)f complex from the thermophilic cyanobacterium, Mastigocladus laminosus, in the presence of the electrochemically positive (p)-side quinone analogue inhibitor, 2,5-dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB). One DBMIB binding site was found. This site is peripheral to the quinone binding space defined by the binding sites of other p-side inhibitors previously resolved in cytochrome bc(1)/b(6)f complexes. This high-affinity site resides in a p-side interfacial niche bounded by cytochrome f, subunit IV, and cytochrome b(6), is close (8 A) to the p-side heme b, but distant (19 A) from the [2Fe-2S] cluster. No significant electron density associated with the DBMIB was found elsewhere in the structure. However, the site at which DBMIB can inhibit light-induced redox turnover is within a few A of the [2Fe-2S] cluster, as shown by the absence of inhibition in mutants of Synechococcus sp. PCC 7002 at iron sulfur protein-Leu-111 near the cluster. The ability of a minimum amount of initially oxidized DBMIB to inhibit turnover of WT complex after a second light flash implies that there is a light-activated movement of DBMIB from the distal peripheral site to an inhibitory site proximal to the [2Fe-2S] cluster. Together with the necessary passage of quinone/quinol through the small Q(p) portal in the complex, it is seen that transmembrane traffic of quinone-like molecules through the core of cytochrome bc complexes can be labyrinthine.     

Calvin cycle multienzyme complexes are bound to chloroplast thylakoid membranes of higher plants in situ

Further evidence is provided that the Calvin cycle enzymes ribose-5-phosphate isomerase (EC 5.3.1.6), ribulose-5-phosphate kinase (Ru-5-P-K, EC 2.7.1.19), ribulose-1,5-bisphosphate carboxylase (RuP2Case, EC 4.1.1.39), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12), sedoheptulose-1,7-bisphosphatase (Sed-1,7-bPase, EC 3.1.3.37), and electron transport protein ferredoxin-NADP+ reductase (FNR, EC 1.18.1.1) are organized into stable CO2-fixing multienzyme complexes with a molecular mass of 900 kDa. Limited trypsinolysis combined with immunoblotting revealed that all of chloroplast stromal Ru-5-P-K and GAPDH is located in enzyme complexes. The Calvin cycle enzyme complexes remain intact indefinitely at lower ionic strength but dissociate into components at KCl concentrations >250 mM. Immunoelectron microscopy showed that Ru-5-P-K, GAPDH, Sed-1,7-bPase, and FNR are bound to stroma-faced thylakoid membranes in situ, whereas RuP2Case and RuP2Case activase are randomly distributed throughout chloroplasts. The results indicate that membrane-bound enzyme supercomplexes may play an important role in photosynthesis.     

Lithium dodecyl sulfate/polyacrylamide gel electrophoresis of thylakoid membranes at 4 degrees C: Characterizations of two additional chlorophyll a-protein complexes

Lithium dodecyl sulfate/polyacrylamide gel electrophoresis of Chlamydomonas reinhardtii thylakoid membranes at room temperature gave two chlorophyll-protein complexes, CP I and CP II, as had been reported previously. However, when the electrophoresis was performed at 4°C, there was an increase in the amount of chlorophyll associated with CP I and CP II, and in addition, three other chlorophyll-protein complexes appeared. Two of these complexes, designated CP III and CP IV, were characterized and found to be similar in their compositions. Each complex contains four to five molecules of chlorophyll a, one molecule of β-carotene, and one polypeptide chain. The apoprotein of CP III is polypeptide 5 (M_r 50,000) and that of CP IV is polypeptide 6 (M_r 47,000); the two polypeptides are structurally unrelated. Chlorophyll-protein complexes similar to C. reinhardtii CP III and CP IV were also detected in higher plants (e.g., Pisum sativum). The apoproteins of the higher plant complexes are immunochemically related to those of the C. reinhardtii complexes, as shown by crossed immunoelectrophoresis. Absorption spectra of CP III and CP IV at -196°C revealed a component at 682 nm. This observation, together with the previous results on photosystem II mutants [Chua, N.-H. & Bennoun, P. (1975) Proc. Natl. Acad. Sci. USA 72, 2175-2179], provides indirect evidence that CP III and CP IV may be involved in the primary photochemistry of photosystem II.     

A defined protein-detergent-lipid complex for crystallization of integral membrane proteins: The cytochrome b6f complex of oxygenic photosynthesis

The paucity of integral membrane protein structures creates a major bioinformatics gap, whose origin is the difficulty of crystallizing these detergent-solubilized proteins. The problem is particularly formidable for hetero-oligomeric integral membrane proteins, where crystallization is impeded by the heterogeneity and instability of the protein subunits and the small lateral pressure imposed by the detergent micelle envelope that surrounds the hydrophobic domain. In studies of the hetero (eight subunit)-dimeric 220,000 molecular weight cytochrome b(6)f complex, derived from the thermophilic cyanobacterium, Mastigocladus laminosus, crystals of the complex in an intact state could not be obtained from highly purified delipidated complex despite exhaustive screening. Crystals of proteolyzed complex could be obtained that grew very slowly and diffracted poorly. Addition to the purified lipid-depleted complex of a small amount of synthetic nonnative lipid, dioleolyl-phosphatidylcholine, resulted in a dramatic improvement in crystallization efficiency. Large crystals of the intact complex grew overnight, whose diffraction parameters are as follows: 94% complete at 3.40 A spacing; R(merge) = 8.8% (38.5%), space group, P6(1)22; and unit cell parameters, a = b = 156.3 A, c = 364.0 A, alpha = beta = 90 degrees, gamma = 120 degrees.It is proposed that the methodology of augmentation of a well-defined lipid-depleted integral membrane protein complex with synthetic nonnative lipid, which can provide conformational stability to the protein complex, may be of general use in the crystallization of integral membrane proteins.     

Electron flow through plastoquinone and cytochromes b6 and f in chloroplasts.

With dark-adapted chloroplasts in which the plastoquinone was oxidized, a partial reduction of cytochrome b6 was obtained upon illumination with a pair of short saturating flashes. The second flash of the pair was much more effective than the first, and the reduction was inhibited by the system II inhibitor diuron. When the plastoquinone pool was reduced, both the reduction and the oxidation of cytochrome b6 were accelerated. The cytochrome b6 oxidation appeared to proceed in association with the reduction of cytochrome f, although these cytochromes are not simply connected in series. From these observations it is inferred that electron flow to the secondary donors of system I alternately caused the reduction and the oxidation of cytochrome b6. An interpretation is offered that also accounts for the transmembrane proton translocation that is associated with the oxidation of plastohydroquinone.     

Release of oxidized plastocyanin from photosystem I limits electron transfer between photosystem I and cytochrome b6f complex in vivo

We used fast absorbance spectroscopy to investigate in vivo binding dynamics and electron transfer between plastocyanin (pc) and photosystem I (PSI), and cytochrome (cyt) f oxidation kinetics in Chlamydomonas reinhardtii mutants in which either the binding or the release of pc from PSI was diminished. Under single flash-excitation conditions, electron flow between PSI and the cyt complex was not affected by a 5-fold lowering of the binding affinity of pc to PSI, as induced by a mutation replacing the tryptophan-651 of the PsaA subunit by a serine residue (PsaA-W651S). On the other hand, electron flow from PSI to the cyt b(6)f complex was very sensitive to a 2- to 3-fold decrease in the rate of pc release from PSI, obtained by replacing the glutamic acid residue 613 of the PsaB subunit with glutamine (PsaB-E613N). Thus, our data indicate that under these experimental conditions the release of oxidized pc limits electron transfer between cyt b(6)f complex and PSI in vivo.     

Lateral diffusion of lipids in complex biological membranes.

Lateral diffusion of lipids in biological membranes may be influenced by polypeptides, proteins, and other nonlipid membrane constituents. Using concepts from scaled-particle theory, we extend the free-volume model for lipid diffusion to membranes having an arbitrarily large number of components. This theory clarifies the interpretation of the free-volume theory, better reproduces the free-area dependence of lipid lateral diffusion rates, and quantitatively predicts the experimental observation that the lateral diffusion rates of membrane lipids are significantly reduced when proteins or polypeptides are incorporated in the membrane.     

Lateral hapten mobility and immunochemistry of model membranes.

A study has been made of the interaction of specific anti-nitroxide (anti-spin label) IgG antibodies, Fab fragments, complement, and liposomes containing dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, and dipalmitoylphosphatidylcholine plus 0-50 mole % cholesterol, together with various concentrations of a head group spin-labeled phospholipid (0.5-0.01 mole % in the plane of the membrane). The spin-labeled lipid is the amide formed from the reaction of an iodoacetamide spin label, N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)iodoacetamide, with dipalmitoylphosphatidylethanolamine. We have reached two conclusions: (i) complement fixation depends on the lateral mobility of the lipid hapten at low hapten concentrations in the plane of the membrane (less than or equal 0.1 mole %) and does not depend strongly on this mobility at high hapten concentrations; (ii) cholesterol may have two distinct effects on complement fixation, one arising from an enhancement of hapten exposure to antibody binding sites and a second due to a modulation of membrane fluidity.     

Mechanism of electron transfer in the cytochrome b/f complex of algae: evidence for a semiquinone cycle

The most widely accepted mechanism of electron and proton transfer within the cytochrome (Cyt) b/f complex derives from the Q-cycle hypothesis originally proposed for the mitochondrial Cyt b/c1 complex by Mitchell [Mitchell, P. (1975) FEBS Lett. 57, 135-137]. In chloroplasts, the Cyt b/f complex catalyzes the oxidation of a plastoquinol at a site, Qo (the plastoquinol binding site), close to the inner aqueous phase and the reduction of a quinone at a site, Qi (the plastoquinone binding site), close to the stromal side of the membrane. In an alternative model, the semiquinone cycle [Wikström, M. & Krab, K. (1986) J. Bioenerg. Biomembr. 18, 181-193], a charged semiquinone formed at site Qo is transferred to site Qi where it is reduced into quinol. Flash-induced kinetics of the redox changes of Cyt b and of the formation of a transmembrane potential have been measured in Chlorella sorokiniana cells incubated in reducing conditions that induce a full reduction of the plastoquinone pool. The experiments were performed in the presence of an uncoupler that collapses the permanent electrochemical proton gradient and thus accelerates the rate of the electrogenic processes. The results show that the electrogenic reaction driven by the Cyt b/f complex precedes the processes of reduction or oxidation of the b-hemes. This electrogenic process is probably due to a transmembrane movement of a charged semiquinone, in agreement with the semiquinone-cycle hypothesis. This mechanism may represent an adaptation to reducing conditions when no oxidized quinone is available at the Qi site.     

Lateral opening of a translocon upon entry of protein suggests the mechanism of insertion into membranes

The structure of the protein-translocating channel SecYEβ from Pyrococcus furiosus at 3.1-Å resolution suggests a mechanism for chaperoning transmembrane regions of a protein substrate during its lateral delivery into the lipid bilayer. Cytoplasmic segments of SecY orient the C-terminal α-helical region of another molecule, suggesting a general binding mode and a promiscuous guiding surface capable of accommodating diverse nascent chains at the exit of the ribosomal tunnel. To accommodate this putative nascent chain mimic, the cytoplasmic vestibule widens, and a lateral exit portal is opened throughout its entire length for partition of transmembrane helical segments to the lipid bilayer. In this primed channel, the central plug still occludes the pore while the lateral gate is opened, enabling topological arbitration during early protein insertion. In vivo, a 15 amino acid truncation of the cytoplasmic C-terminal helix of SecY fails to rescue a secY-deficient strain, supporting the essential role of this helix as suggested from the structure.     

Sequence homology and structural similarity between cytochrome b of mitochondrial complex III and the chloroplast b6-f complex: position of the cytochrome b hemes in the membrane.

The amino acid sequences of cytochrome b of complex III from five different mitochondrial sources (human, bovine, mouse, yeast, and Aspergillus nidulans) and the chloroplast cytochrome b6 from spinach show a high degree of homology. Calculation of the distribution of hydrophobic residues with a "hydropathy" function that is conserved in this family of proteins implies that the membrane-folding pattern of the 42-kilodalton (kDa) mitochondrial cytochromes involves 8-9 membrane-spanning domains. The smaller 23-kDa chloroplast cytochrome appears to fold in five spanning domains that are similar to the first five of the mitochondria. Four highly conserved histidines are considered to be the likely ligands for the two hemes. The positions of the histidines along the spanning segments and in a cross section of the membrane-spanning alpha helices implies that two ligand pairs, His-82-His-197/198 and His-96-His-183, bridge the spanning peptides II and V, and the two hemes reside on opposite sides of the hydrophobic membrane core. In addition, the 17-kDa protein of the chloroplast b6-f complex appears to contain one or more of the functions of the COOH-terminal end of the mitochondrial cytochrome b polypeptide.     

Erythrocyte membranes undergo cooperative, pH-sensitive state transitions in the physiological temperature range: evidence from Raman spectroscopy.

We have examined the Raman scattering from erythrocyte ghosts at 2700 to 3000 cm-1 (CH-stretching region). Plots of the intensity (I) of the 2930 cm-1 band relative to the intensity of the thermally stable 2850 cm-1 band, i.e., the [I2930/I2850] ratio, as a function of temperature reveal a sharp discontinuity, which at pH 7.4 has a lower limit of 38 degrees and is irreversible above 42 degrees. [I2930/I2850] is stable between pH 7.0 and pH 7.4, but increases or decreases sharply below pH 7.0 or above pH 7.5, respectively. Reduction of pH to 6.5 lowers the transition temperature by about 16 degrees, and a shift to pH 6.0 drops the transition range to 0 to 7 degrees. The above effects of temperature and pH on Raman scattering closely correspond to those detected by studies on the interaction of membrane protein fluorophores and lipid-soluble fluorescence quenchers [Bieri, V. and Wallach, D.F.H. (1975) Biochim. Biophys. Acta 406, 415-423]. Taken together, these results suggest that the transitions represent concerted process, involving hydrophobic amino acid residues and lipid chains at apolar protein-lipid boundaries.     

Resolution of cytochrome oxidase into two component complexes.

Cytochrome c oxidase (ferrocytochrome c: oxygen oxidoreductase, EC 1.9.3.1) has been resolved into a pair of complexes of unequal molecular weight. The larger complex (electron transfer complex) contains exclusively the oxidation-reduction proteins characteristic of cytochrome oxidase; the smaller complex (ion transfer complex) shows exclusively the capability for cation-dependent induction of the fluorescence of 8-anilino-1-naphthalenesulfonic acid--a capability demonstrable in preparations of cytochrome oxidase. The duplex nature of cytochrome oxidase has important implications for the mechanism of energy coupling.     

Increase in apparent compressibility of cytochrome c upon oxidation.

The apparent molal adiabatic compressibilities of ferri- and ferrocytochrome c have been determined from measurements of density and sound velocity. The values found were +2.99 X 10(-8) and -2.40 X 10(-8) cm5 mol-1 dyne-1 for the ferri and ferro forms, respectively. Experiments were performed on identical solutions containing either the oxidized or reduced form of protein. Solutions of ferricytochrome c were found to have significantly greater adiabatic compressibility than equivalent solutions of ferrocytochrome c at 25 degrees C and pH 7.15. The remarkable similarity of the three-dimensional structures of the ferri and ferro proteins [Takano, T. & Dickerson, R.E. (1980) Proc. Natl. Acad. Sci. USA 77, 6371-6375] strongly suggests that this difference in compressibility is due to an increase in volume fluctuations within ferricytochrome c relative to the ferro form rather than a change in equilibrium structure or hydration. Such a difference in the dynamic properties of the structures is consistent with both the crystallographic thermal B factors and the observed increase in amide hydrogen exchange kinetics when ferrocytochrome c is oxidized. The relative magnitude of the root mean square volume fluctuations is approximated from an ideal solution treatment of the compressibility data and yields a ratio of delta Vrms (ferri cyt c)/ delta Vrms (ferro cyt c) = 1.3.     

Direct observation of large chiral domains in chloroplast thylakoid membranes by differential polarization microscopy.

Long-range chiral organization of the pigment-protein complexes in mature granal chloroplasts has been established by differential polarization imaging and local circular dichroism spectra. Linear and circular dichroism images of oriented chloroplasts were obtained in a confocal differential polarization microscope. The circular dichroism images display signals of opposite signs emerging from discrete regions with local dichroic values much larger than anticipated, indicating domains in the thylakoid membranes having long-range chiral organization. These domains are associated with positive and negative circular dichroism bands obtained at specific locations on the chloroplasts. Surprisingly, the local circular dichroism spectra do not display the excitonic shape of spectra obtained for macroscopic suspensions, but the latter can be produced by superposition of two local spectra of opposite sign. These data are evidence for the existence of long-range chiral order of the pigment-protein complexes in thylakoid membranes. The possible role of the long-range chiral domains in the efficiency of energy delocalization through the thylakoid membranes is discussed.     

Evidence that two forms of bovine erythrocyte cytochrome b5 are identical to segments of microsomal cytochrome b5.

Homogeneous preparations of two forms of soluble cytochrome b5 have been obtained from bovine erythrocytes by successive chromatography on DEAE-cellulose, Bio-Gel P-60, and DEAE-Sephadex. Although the two forms could be separated on disc gel electrophoresis, they appeared to have similar molecular weights of approximately 12,000 and identical visible absorbance spectra. The tryptic hemepeptides derived from the two forms of bovine erythrocyte cytochrome b5 are electrophoretically indistinguishable from each other and from the tryptic core hemepeptide derived from liver microsomal cytochrome b5. The bovine erythrocyte tryptic hemepeptide was purified to homogeneity; its amino acid composition was shown to be identical to that of tryptic hemepeptide from liver microsomal cytochrome b5. The amino acid compositions of the two isolatable forms of erythrocyte cytochrome b5 correspond well to the compositions of the 97- and 95-residue segments of native liver microsomal cytochrome b5 that begin at the NH2 terminus. These results agree with the hypothesis that soluble erythrocyte cytochrome b5 is derived from microsomal protein by proteolysis during erythroid maturation.