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Membrane association is required for cell transformation by pp60v-src (v-Src), the product of the v-src oncogene of Rous sarcoma virus. Previous experiments have identified two NH2-terminal membrane-binding motifs: a myristate (14-carbon acyl chain) attached to the NH2-terminal glycine and three basic residues at positions 5, 7, and 9 of Src. We examined the membrane binding of each motif using myristylated (myr-src) and nonmyristylated (nonmyr-src) peptides corresponding to the NH2 terminus of Src. All myristylated peptides partitioned equally well onto electrically neutral phosphatidylcholine vesicles (K1 = 10(4) M-1). Identical binding has been observed for simple myristylated peptides (e.g., myr-Gly) and arises from the hydrophobic insertion of the myristate into the bilayer. A nonmyristylated peptide corresponding to residues 2-16 of Src [nonmyr-src(2-16), net charge = +5] bound to vesicles containing 33% monovalent acidic phospholipids with K1 = 10(3) M-1. Penta(lysine) (+5 net charge) exhibits the same binding behavior, which is due to the electrostatic interaction between basic residues and acidic lipids. The corresponding myristylated peptide, myr-src(2-16), binds 3 orders of magnitude more strongly to vesicles containing 33% acidic lipids than to neutral vesicles. The resulting apparent association constant, K1 = 10(7) M-1, is approximately equal to the product of the partition coefficients for the two individual interactions. This 10(7) M-1 binding is sufficiently strong to anchor the Src protein to biological membranes. We propose a simple model that explains the observed synergism between the two peptide-membrane interactions.

Cytochrome b559 (cyt b559) is an intrinsic and essential component of the photosystem II (PSII) protein complex, but its function, stoichiometry, and electron-transfer kinetics in the physiological system are not well-defined. In this study, we have used flash-detection optical spectroscopy to measure the kinetics and yields of photooxidation and dark reduction of cyt b559 in untreated, O2-evolving PSII-enriched membranes at room temperature. The dark redox states of cyt b559 and the primary electron acceptor, QA, were determined over the pH range 5.0-8.5. Both the fraction of dark-oxidized cyt b559 and dark-reduced QA increased with increasing acidity. Consistent with these results, an acid-induced drop in pH from 8.5 to 4.9 in a dark-adapted sample caused the oxidation of cyt b559, indicating a shift in the redox state during the dark reequilibration. As expected from the dark redox state of cyt b559, the rate and extent of photooxidation of cyt b559 during continuous illumination decreased toward more acidic pH values. After a single, saturating flash, the rate of photooxidation of cyt b559 was of the same order of magnitude as the rate of S2QA- charge recombination. In untreated PSII samples at pH 8.0 with 42% of cyt b559 oxidized and 15% of QA reduced in the dark, 4.7% of one copy of cyt b559 was photooxidized after one flash with a t1/2 of 540 +/- 90 ms. On the basis of our previous work [Buser, C. A., Thompson, L. K., Diner, B. A., & Brudvig, G. W (1990) Biochemistry 29, 8977] and the data presented here, we conclude that Sn+1, YZ., and P680+ are in redox equilibrium and cyt b559 (and YD) are oxidized via P680+. After a period of illumination sufficient to fully reduce the plastoquinone pool, we also observed the pH-dependent dark reduction of photooxidized cyt b559, where the rate of reduction decreased with decreasing pH and was not observed at pH < 6.4. To determine the direct source of reductant to oxidized cyt b559, we studied the dark reduction of cyt b559 and the reduction of the PQ pool as a function of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) concentration. We find that DCMU inhibits the reduction of cyt b559 under conditions where the plastoquinone pool and QA are reduced. We conclude that QB-. (H+) or QBH2 is the most likely source of the electron required for the reduction of oxidized cyt b559.(ABSTRACT TRUNCATED AT 400 WORDS)

We have used flash-detection optical and electron paramagnetic resonance spectroscopy to measure the kinetics and yield per flash of the photooxidation of cytochrome b559 and the yield per flash of the photooxidation of the tyrosine residue YD in Mn-depleted photosystem II (PSII) membranes at room temperature. The initial charge separation forms YZ+ QA-. Following this, cytochrome b559 is oxidized on a time scale of the same order and with the same pH dependence as is observed for the decay of YZ+; under the conditions of our experiments, the decay of YZ+ is determined by the lifetime of YZ+ QA-. In order to explain this observation, we have constructed a model for electron donation in which YZ+ and P680+ are in redox equilibrium and cytochrome b559 and YD are oxidized via P680+. Using our results, together with data from earlier investigations of the kinetics of electron transfer from YZ to P680+ and charge recombination of YZ+ QA-, we have obtained the first global fit for electron donation in Mn-depleted PSII that accounts for the data over the pH range from 5 to 7.5. From these calculations, we have obtained the intrinsic rate constants of all the electron-donation reactions in Mn-depleted PSII. These rate constants allow us to calculate the free energy difference between YZ+ P680 and YZ P680+, which is found to increase by 47 +/- 4 mV/pH from pH 5 to 6 and is observed to increase more slowly per pH unit for pH greater than 6. An important conclusion of our experimental work is that the rates of photooxidation of cytochrome b559 and YD are determined by the lifetime of the oxidizing equivalent on YZ/P680. Extension of our model to oxygen-evolving PSII samples leads to the prediction that the kinetics and yields of electron donation from cytochrome b559 and YD to P680+ will depend on the S2- or S3-state lifetime.

The binding of Src to phospholipid membranes requires both hydrophobic insertion of its myristate into the hydrocarbon interior of the membrane and nonspecific electrostatic interaction of its N-terminal cluster of basic residues with acidic phospholipids. We provide a theoretical description of the electrostatic partitioning of Src onto phospholipid membranes. Specifically, we use molecular models to represent a nonmyristoylated peptide corresponding to residues 2-19 of Src [nonmyr-Src(2-19); GSSKSKPKDPSQRRRSLE-NH2] and a phospholipid bilayer, calculate the electrostatic interaction by solving the nonlinear Poisson-Boltzmann equation, and predict the molar partition coefficient using statistical thermodynamics. The theoretical predictions agree with experimental data obtained by measuring the partitioning of nonmyr-Src(2-19) onto phospholipid vesicles: membrane binding increases as the mole percent of acidic lipid in the vesicles is increased, the ionic strength of the solution is decreased, or the net positive charge of the peptide is increased. The theoretical model also correctly predicts the measured partitioning of the myristoylated peptide, myr-Src(2-19); for example, adding 33% acidic lipid to electrically neutral vesicles increases the partitioning of myr-Src(2-19) 100-fold. Phosphorylating either serine 12 (by protein kinase C) or serine 17 (by cAMP-dependent protein kinase) decreases the partitioning of myr-Src(2-19) onto vesicles containing acidic lipid 10-fold. We investigated the effect of phosphorylation on the localization of Src to biological membranes by expressing fusion constructs of Src's N terminus with a soluble carrier protein in COS-1 cells; phosphorylation produces a small shift in the distribution of the Src chimeras from the plasma membrane to the cytosol.

The stoichiometry of cytochrome b559 (one or two copies) per reaction center of photosystem II (PSII) has been the subject of considerable debate. The molar ratio of cytochrome b559 has a number of significant implications on our understanding of the functional role of cytochrome b559, the mechanism of electron donation in PSII, and the stoichiometry of the other redox-active, reaction center components. We have reinvestigated the stoichiometry of cytochrome b559 in PSII-enriched and thylakoid membranes, using differential absorbance and electron paramagnetic resonance spectroscopies. The data from both quantitation procedures strongly indicate only one copy of cytochrome b559 per reaction center in PSII-enriched membranes and also suggest one copy of cytochrome b559 per reaction center in thylakoid membranes.

Cytochrome b559 is an essential component of the photosystem II (PSII) protein complex. Its function, which has long been an unsolved puzzle, is likely to be related to the unique ability of PSII to oxidize water. We have used EPR spectroscopy and spectrophotometric redox titrations to probe the structure of cytochrome b559 in PSII samples that have been treated to remove specific components of the complex. The results of these experiments indicate that the low-temperature photooxidation of cytochrome b559 does not require the presence of the 17-, 23-, or 33-kDa extrinsic polypeptides or the Mn complex (the active site in water oxidation). We observe a shift in the g value of the EPR signal of cytochrome b559 upon warming a low-temperature photooxidized sample, which presumably reflects a change in conformation to accommodate the oxidized state. At least three redox forms of cytochrome b559 are observed. Untreated PSII membranes contain one high-potential (375 mV) and one intermediate-potential (230 mV) cytochrome b559 per PSII. Thylakoid membranes also appear to contain one high-potential and one intermediate-potential cytochrome b559 per PSII, although this measurement is more difficult due to interference from other cytochromes. Removal of the 17- and 23-kDa extrinsic polypeptides from PSII membranes shifts the composition to one intermediate-potential (170 mV) and one low-potential (5 mV) cytochrome b559. This large decrease in potential is accompanied by a very small g-value change (0.04 at gz), indicating that it is the environment and not the ligand field of the heme which changes significantly upon the removal of the 17- and 23-kDa polypeptides.