NMR analysis of the transient complex between membrane photosystem I and soluble cytochrome c6

A structural analysis of the surface areas of cytochrome c(6), responsible for the transient interaction with photosystem I, was performed by NMR transverse relaxation-optimized spectroscopy. The hemeprotein was titrated by adding increasing amounts of the chlorophyllic photosystem, and the NMR spectra of the free and bound protein were analyzed in a comparative way. The NMR signals of cytochrome c(6) residues located at the hydrophobic and electrostatic patches, which both surround the heme cleft, were specifically modified by binding. The backbones of internal residues close to the hydrophobic patch of cytochrome c(6) were also affected, a fact that is ascribed to the conformational changes taking place inside the hemeprotein when interacting with photosystem I. To the best of our knowledge, this is the first structural analysis by NMR spectroscopy of a transient complex between soluble and membrane proteins.     

A large fraction of PsaF is nonfunctional in photosystem I complexes lacking the PsaJ subunit

PsaJ is a small hydrophobic subunit of the photosystem I complex (PSI) whose function is not yet fully understood. Here we describe mutants of the green alga Chlamydomonas reinhardtii, in which the psaJ chloroplast gene has been inactivated either in a wild-type or in a PsaF-deficient nuclear background. Cells lacking one or both subunits grow photoautotrophically and contain normal levels of PSI. Flash-absorption spectroscopy performed with isolated PSI particles isolated from the PsaJ-deficient strain indicates that only 30% of the PSI complexes oxidize plastocyanin (Pc) or cytochrome c6 (Cyt c6) with kinetics identical to wild type, whereas the remaining 70% follow slow kinetics similar to those observed with PsaF-deficient PSI complexes. This feature is not due to partial loss of PsaF, as the PsaJ-less PSI complex contains normal levels of the PsaF subunit. The N-terminal domain of PsaF can be cross-linked to Pc and Cyt c6 indicating that in the absence of PsaJ, this domain is exposed in the lumenal space. Therefore, the decreased amount of functional PsaF revealed by the electron-transfer measurements is best explained by a displacement of the N-terminal domain of PsaF which is known to provide the docking site for Pc and Cyt c6. We propose that one function of PsaJ is to maintain PsaF in a proper orientation which allows fast electron transfer from soluble donor proteins to P700(+).     

Thioredoxin-mediated reduction of the photosystem I subunit PsaF and activation through oxidation by the interaction partner plastocyanin

In the photosynthetic electron-transfer chain, the photosystem I subunit PsaF is involved in the specific binding of plastocyanin. Using fluorescence electrophoresis we show here that the luminal domain of PsaF is a target for thioredoxin-mediated reduction of the Cys residues 8 and 63. Furthermore, by using NMR spectroscopy, we show that the thiolated form of PsaF has a lower affinity towards reduced plastocyanin than when the disulfide bridge is intact. Time-resolved absorbance measurements and fluorescence electrophoresis shows that oxidized plastocyanin can re-oxidize PsaF and thus restore the active form.     

Identification of the plastocyanin binding subunit of photosystem I

Plastocyanin is specifically cross-linked by incubation with N-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) to a subunit of photosystem I in stroma lamellae and in isolated photosystem I complex. SDS-PAGE shows the disappearance of a 18.5 kDa subunit and the appearance of a new 31.5 kDa protein which was recognized by anti-plastocyanin antibodies. The isolated subunit was identified by its N-terminal amino acid sequence as the mature peptide coded by the nuclear gene psaF [Steppuhn et al. (1988) FEBS Lett. 237, 218–224]. P700⁺ was reduced by cross-linked plastocyanin with the same halftime of 13 μs as found in the native complex. This is evidence that cross-linking conserved the orientation of the complex and that the 18.5 kDa subunit provides the conformation of photosystem I necessary for the extremely rapid electron transfer from plastocyanin to P700⁺.     

Identification of mosquito bloodmeals using mitochondrial cytochrome oxidase subunit I and cytochrome b gene sequences

Abstract.  Primer pairs were designed and protocols developed to selectively amplify segments of vertebrate mitochondrial cytochrome oxidase subunit 1 (COI) and cytochrome b (Cyt b ) mtDNA from the bloodmeals of mosquitoes (Diptera: Culicidae). The protocols use two pairs of nested COI primers and one pair of Cyt b primers to amplify short segments of DNA. Resultant sequences are then compared with sequences in GenBank, using the BLAST function, for putative host identification. Vertebrate DNA was amplified from 88% of our sample of 162 wild-caught, blood-fed mosquitoes from Oregon, U.S.A. and GenBank BLAST searches putatively identified 98% of the amplified sequences, including one amphibian, seven mammalian and 14 avian species. Criteria and caveats for putative identification of bloodmeals are discussed.     

Automated identification of SUMOylation sites using mass spectrometry and SUMmOn pattern recognition software

Tandem mass spectrometry (MS/MS) allows for the rapid identification of many types of post-translational modifications (PTMs), especially those that can be detected by a diagnostic mass shift in one or more peptide fragment ions (for example, phosphorylation). But some PTMs (for example, SUMOs and other ubiquitin-like modifiers) themselves produce multiple fragment ions; combined with fragments from the modified target peptide, a complex overlapping fragmentation pattern is thus generated, which is uninterpretable by standard peptide sequencing software. Here we introduce SUMmOn, an automated pattern recognition tool that detects diagnostic PTM fragment ion series within complex MS/MS spectra, to identify modified peptides and modification sites within these peptides. Using SUMmOn, we demonstrate for the first time that human SUMO-1 multimerizes in vitro primarily via three N-terminal lysines, Lys7, Lys16 and Lys17. Notably, our method is theoretically applicable to any type of modification or chemical moiety generating a unique fragment ion pattern.     

Identification of three phosphorylation sites in the alpha7 subunit of the yeast 20S proteasome in vivo using mass spectrometry

The 26S proteasome complex, which consists of a 20S proteasome and a pair of 19S regulatory particles, plays important roles in the degradation of ubiquitinated proteins in eukaryotic cells. The alpha7 subunit of the budding yeast 20S proteasome is a major phosphorylatable subunit; serine residue(s) in its C-terminal region are phosphorylated in vitro by CKII. However, the exact in vivo phosphorylation sites have not been identified. In this study, using electrospray ionization quadrupole time-of-flight mass spectrometry analysis, we detected a mixture of singly, doubly, and triply phosphorylated C-terminal peptides isolated from a His-tagged construct of the alpha7 subunit by nickel-immobilized metal affinity chromatography. In addition, we identified three phosphorylation sites in the C-terminal region using MS/MS analysis and site-directed mutagenesis: Ser258, Ser263, and Ser264 residues. The MS/MS analysis of singly phosphorylated peptides showed that phosphorylation at these sites did not occur successively.     

Electron transfer between liposomal cytochrome c1 and cytochrome c: catalytic implications of electrostatic potentials

Kinetics measurements of the electron transfer between ferricytochrome c and liposomal ferrocytochrome c1 (with and without the hinge protein) were performed. The observed rate constants(kobs) of electron transfer between liposomal ferrocytochrome c1 and ferricytochrome c at different ionic strengths were measured in cacodylate buffer, pH 7.4, at 2 C. The effect of ionic strength on the rate constant(kobs) of electron transfer between liposomal cytochrome c1 and cytochrome c is far greater than that in the solution kinetics (Kim, C.H., Balny, C. and King, T.E. (1987) J. Biol. Chem. 262, 8103-8108). The result demonstrates that the membrane bound cytochrome c1 creates a polyelectrolytic microenvironment which appears to be involved in the control of electron transfer and can be modulated by the ionic strength. The involvement of electrostatic potentials in the electron transfer between the membrane bound cytochrome c1 and cytochrome c is discussed in accord with the experimental results and a polyelectrolyte theory.     

Identification of the structural gene for yeast cytochrome c oxidase subunit I on mitochondrial DNA.

Mitochondrial protein synthesis was analyzed in the yeast mit^? mutants of $\text{Saccharomyces}$$\text{cerevisiae}$ which specifically lack cytochrome $\text{c}$ oxidase. [³H]leucine labeled polypeptides synthesized in yeast OXI 3 mutant were analyzed by means of immunoprecipitation and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). When compared to control, subunit I was not detectable. This result was substantiated by growing OXI 3 mutant in the presence of cycloheximide, an inhibitor of cytoplasmic protein synthesis. Under such conditions SDS-PAGE analysis of [³H]leucine labeled immunoprecipitate shows the absence of subunit I. These data show that the OXI 3 locus contains the structural gene for cytochrome $\text{c}$ oxidase subunit I.     

Identification of Channa species using the partial cytochrome c oxidase subunit I (COI) gene as a DNA barcoding marker

The snakehead fish of the genus Channa are an important food fish in China. However, the molecular identification and phylogeny of this genus is poorly understood. Here, we present the utility of partial sequences of the COI gene for use in DNA barcoding for the identification of Channa individuals, which includes four species: Channa argus, Channa maculata, Channa asiatica, and Channa striata. A total of 19 haplotypes were identified in this study. The interspecific K2P distances were higher than intraspecific distances. The lowest interspecific distance (0.091) was between C. argus and C. maculata while the highest interspecific distance (0.219) was between C. argus and C. striata. No intraspecific–interspecific distance overlaps were observed, and a distinct barcoding gap was found between intraspecific and interspecific distances in each species. Our results showed that the partial COI gene is an effective DNA barcoding marker for identifying Channa species.     

Identification of RET autophosphorylation sites by mass spectrometry

The catalytic and signaling activities of RET, a receptor-type tyrosine kinase, are regulated by the autophosphorylation of several tyrosine residues in the cytoplasmic region of RET. Some studies have revealed a few possible autophosphorylation sites of RET by [(32)P]phosphopeptide mapping or by using specific anti-phosphotyrosine antibodies. To ultimately identify these and other autophosphorylation sites of RET, we performed mass spectrometry analysis of an originally prepared RET recombinant protein. Both the autophosphorylation and kinase activity of myelin basic protein as an external substrate of the recombinant RET protein were substantially elevated in the presence of ATP without stimulation by a glial cell line-derived neurotrophic factor, a natural ligand for RET. Mass spectrometric analysis revealed that RET Tyr(806), Tyr(809), Tyr(900), Tyr(905), Tyr(981), Tyr(1062), Tyr(1090), and Tyr(1096) were autophosphorylation sites. Levels of autophosphorylation and kinase activity of RET-MEN2A (multiple endocrine neoplasia 2A), a constitutively active form of RET with substitution of Tyr(900) by phenylalanine (Y900F), were comparable with those of original RET-MEN2A, whereas those of the mutant Y905F were greatly decreased. Interestingly, those of a double mutant, Y900F/Y905F, were completely abolished. Both the kinase activity and transforming activity were impaired in the mutants Y806F and Y809F. These results provide convincing evidence for both previously suggested and new tyrosine autophosphorylation sites of RET as well as for novel functions of Tyr(806), Tyr(809), and Tyr(900) phosphorylation in both catalytic kinase activities and cell growth. The significance of the identified autophosphorylation sites in various protein-tyrosine kinases registered in a data base is discussed in this paper.     

The PSI-K subunit of photosystem I is involved in the interaction between light-harvesting complex I and the photosystem I reaction center core

PSI-K is a subunit of photosystem I. The function of PSI-K was characterized in Arabidopsis plants transformed with a psaK cDNA in antisense orientation, and several lines without detectable PSI-K protein were identified. Plants without PSI-K have a 19% higher chlorophyll a/b ratio and 19% more P700 than wild-type plants. Thus, plants without PSI-K compensate by making more photosystem I. The photosystem I electron transport in vitro is unaffected in the absence of PSI-K. Light response curves for oxygen evolution indicated that the photosynthetic machinery of PSI-K-deficient plants have less capacity to utilize light energy. Plants without PSI-K have less state 1-state 2 transition. Thus, the redistribution of absorbed excitation energy between the two photosystems is reduced. Low temperature fluorescence emission spectra revealed a 2-nm blue shift in the long wavelength emission in plants lacking PSI-K. Furthermore, thylakoids and isolated PSI without PSI-K had 20-30% less Lhca2 and 30-40% less Lhca3, whereas Lhca1 and Lhca4 were unaffected. During electrophoresis under mildly denaturing conditions, all four Lhca subunits were partially dissociated from photosystem I lacking PSI-K. The observed effects demonstrate that PSI-K has a role in organizing the peripheral light-harvesting complexes on the core antenna of photosystem I.     

Unexpected changes in photosystem I function in a cytochrome c6-deficient mutant of the cyanobacterium Synechocystis PCC 6803

Cytochrome c6, the product of the petJ gene, is a photosynthetic electron carrier in cyanobacteria, which transfers electrons to photosystem I and which is synthesised under conditions of copper deficiency to functionally replace plastocyanin. The photosystem I photochemical activity (energy storage, photoinduced P700 redox changes) was examined in a petJ-null mutant of Synechocystis PCC 6803. Surprisingly, photosystem I activity in the petJ-null mutant grown in the absence of copper was not much affected. However, in a medium with a low inorganic carbon concentration and with NH4+ ion as nitrogen source, the mutant displayed growth inhibition. Analysis showed that, especially in the latter, the isiAB operon, encoding flavodoxin and CP43', an additional chlorophyll a antenna, was strongly expressed in the mutant. These proteins are involved in photosystem I function and organisation and are proposed to assist in prevention of overoxidation of photosystem I at its lumenal side and overreduction at its stromal side.     

Characterization of four covalently-linked yeast cytochrome c/cytochrome c peroxidase complexes: Evidence for electrostatic interaction between bound cytochrome c molecules

Four covalent complexes between recombinant yeast cytochrome c and cytochrome c peroxidase (rCcP) were synthesized via disulfide bond formation using specifically designed protein mutants (Papa, H. S., and Poulos, T. L. (1995) Biochemistry 34, 6573-6580). One of the complexes, designated V5C/K79C, has cysteine residues replacing valine-5 in rCcP and lysine-79 in cytochrome c with disulfide bond formation between these residues linking the two proteins. The V5C/K79C complex has the covalently bound cytochrome c located on the back-side of cytochrome c peroxidase, approximately 180 degrees from the primary cytochrome c-binding site as defined by the crystallographic structure of the 1:1 noncovalent complex (Pelletier, H., and Kraut J. (1992) Science 258, 1748-1755). Three other complexes have the covalently bound cytochrome c located approximately 90 degrees from the primary binding site and are designated K12C/K79C, N78C/K79C, and K264C/K79C, respectively. Steady-state kinetic studies were used to investigate the catalytic properties of the covalent complexes at both 10 and 100 mM ionic strength at pH 7.5. All four covalent complexes have catalytic activities similar to those of rCcP (within a factor of 2). A comprehensive study of the ionic strength dependence of the steady-state kinetic properties of the V5C/K79C complex provides evidence for significant electrostatic repulsion between the two cytochromes bound in the 2:1 complex at low ionic strength and shows that the electrostatic repulsion decreases as the ionic strength of the buffer increases.     

The interaction between plastocyanin and photosystem I is inefficient in transgenic Arabidopsis plants lacking the PSI-N subunit of photosystem I

The PSI-N subunit of photosystem I (PSI) is restricted to higher plants and is the only subunit located entirely in the thylakoid lumen. The role of the PSI-N subunit in the PSI complex was investigated in transgenic Arabidopsis plants which were generated using antisense and co-suppression strategies. Several lines without detectable levels of PSI-N were identified. The plants lacking PSI-N assembled a functional PSI complex and were capable of photoautotrophic growth. When grown on agar media for several weeks the plants became chlorotic and developed significantly more slowly. However, under optimal growth conditions, the plants without PSI-N were visually indistinguishable from the wild-type although several photosynthetic parameters were affected. In the transformants, the second-order rate constant for electron transfer from plastocyanin to P700+, the oxidized reaction centre of PSI, was only 55% of the wild-type value, and steady-state NADP+ reduction was decreased to a similar extent. Quantum yield of oxygen evolution and PSII photochemistry were about 10% lower than in the wild-type at leaf level. Photochemical fluorescence quenching was lowered to a similar extent. Thus, the 40-50% lower activity of PSI at the molecular level was much less significant at the whole-plant level. This was partly explained by a 17% increase in PSI content in the plants lacking PSI-N.     

Site-directed mutagenesis of cytochrome c(6) from Anabaena species PCC 7119. Identification of surface residues of the hemeprotein involved in photosystem I reduction

A number of surface residues of cytochrome c(6) from the cyanobacterium Anabaena sp. PCC 7119 have been modified by site-directed mutagenesis. Changes were made in six amino acids, two near the heme group (Val-25 and Lys-29) and four in the positively charged patch (Lys-62, Arg-64, Lys-66, and Asp-72). The reactivity of mutants toward the membrane-anchored complex photosystem I was analyzed by laser flash absorption spectroscopy. The experimental results indicate that cytochrome c(6) possesses two areas involved in the redox interaction with photosystem I: 1) a positively charged patch that may drive its electrostatic attractive movement toward photosystem I to form a transient complex and 2) a hydrophobic region at the edge of the heme pocket that may provide the contact surface for the transfer of electrons to P(700). The isofunctionality of these two areas with those found in plastocyanin (which acts as an alternative electron carrier playing the same role as cytochrome c(6)) are evident.     

Peculiar properties of the PsaF photosystem I protein from the green alga Chlamydomonas reinhardtii: presequence independent import of the PsaF protein into both chloroplasts and mitochondria

It has previously been shown that presequences of nuclear-encoded chloroplast proteins from the green alga Chlamydomonas reinhardtii contain a region that may form an amphiphilic -helix, a structure characteristic of mitochondrial presequences. We have tested two precursors of chloroplast proteins (the PsaF and PsaK photosystem I subunits) from C. reinhardtii for the ability to be imported into spinach leaf mitochondria in vitro. Both precursors bound to spinach mitochondria. The PsaF protein was converted into a protease-protected form with high efficiency in a membrane potential-dependent manner, indicating that the protein had been imported, whereas the PsaK protein was not protease protected. The protease protection of PsaF was not inhibited by a synthetic peptide derived from the presequence of the N. plumbaginifolia mitochondrial F₁ subunit. Furthermore, if the presequence of PsaF was truncated or deleted by in vitro mutagenesis, the protein was still protease-protected with approximately the same efficiency as the full-length precursor. These results indicate that PsaF can be imported by spinach mitochondria in a presequence-independent manner. However, even in the absence of the presequence, this process was membrane potential-dependent. Interestingly, the presequence-truncated PsaF proteins were also protease-protected upon incubation with C. reinhardtii chloroplasts. Our results indicate that the C. reinhardtii chloroplast PsaF protein has peculiar properties and may be imported not only into chloroplasts but also into higher-plant mitochondria. This finding indicates that additional control mechanisms in the cytosol that are independent of the presequence are required to achieve sorting between chloroplasts and mitochondria in vivo.Abbreviations cTP chloroplast transit peptide - mTP mitochondrial targeting peptide - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - pF₁(1,25) a synthetic peptide derived from the first 25 residues of the Nicotiana plumbaginifolia mitochondrial ATP synthase F₁ subunit - PsaF(2–30) and PsaF(2–61) mutant proteins lacking regions corresponding to residues 2–30 and 2–61 in the PsaF precursor protein, respectively     

cAMP-dependent tyrosine phosphorylation of subunit I inhibits cytochrome c oxidase activity

Signaling pathways targeting mitochondria are poorly understood. We here examine phosphorylation by the cAMP-dependent pathway of subunits of cytochrome c oxidase (COX), the terminal enzyme of the electron transport chain. Using anti-phospho antibodies, we show that cow liver COX subunit I is tyrosinephosphorylated in the presence of theophylline, a phosphodiesterase inhibitor that creates high cAMP levels, but not in its absence. The site of phosphorylation, identified by mass spectrometry, is tyrosine 304 of COX catalytic subunit I. Subunit I phosphorylation leads to a decrease of V(max) and an increase of K(m) for cytochrome c and shifts the reaction kinetics from hyperbolic to sigmoidal such that COX is fully or strongly inhibited up to 10 mum cytochrome c substrate concentrations, even in the presence of allosteric activator ADP. To assess our findings with the isolated enzyme in a physiological context, we tested the starvation signal glucagon on human HepG2 cells and cow liver tissue. Glucagon leads to COX inactivation, an effect also observed after incubation with adenylyl cyclase activator forskolin. Thus, the glucagon receptor/G-protein/cAMP pathway regulates COX activity. At therapeutic concentrations used for asthma relief, theophylline causes lung COX inhibition and decreases cellular ATP levels, suggesting a mechanism for its clinical action.     

Identification of the interactions between cytochrome P450 2E1 and cytochrome b5 by mass spectrometry and site-directed mutagenesis

The reaction cycles of cytochrome P450s (P450) require input of two electrons. Electrostatic interactions are considered important driving forces in the association of P450s with their redox partners, which in turn facilitates the transfer of the two electrons. In this study, the cross-linking reagent, 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), was used to covalently link cytochrome P450 2E1 (CYP2E1) with cytochrome b(5) (b(5)) through the formation of specific amide bonds between complementary charged residue pairs. Cross-linked peptides in the resulting protein complex were distinguished from non-cross-linked peptides using an (18)O-labeling method on the basis that cross-linked peptides incorporate twice as many (18)O atoms as non-cross-linked peptides during proteolysis conducted in (18)O-water. Subsequent tandem mass spectrometric (MS/MS) analysis of the selected cross-linked peptide candidates led to the identification of two intermolecular cross-links, Lys(428)(CYP2E1)-Asp(53)(b(5)) and Lys(434)(CYP2E1)-Glu(56)(b(5)), which provides the first direct experimental evidence for the interacting orientations of a microsomal P450 and its redox partner. The biological importance of the two ion pairs for the CYP2E1-b(5) interaction, and the stimulatory effect of b(5), was confirmed by site-directed mutagenesis. Based on the characterized cross-links, a CYP2E1-b(5) complex model was constructed, leading to improved insights into the protein interaction. The described method is potentially useful for mapping the interactions of various P450 isoforms and their redox partners, because the method is relatively rapid and sensitive, and is capable of suggesting not only protein interacting regions, but also interacting orientations.     

The luminal helix l of PsaB is essential for recognition of plastocyanin or cytochrome c6 and fast electron transfer to photosystem I in Chlamydomonas reinhardtii.

At the lumenal side of photosystem I (PSI) in cyanobacteria, algae, and vascular plants, proper recognition and binding of the donor proteins plastocyanin (pc) and cytochrome (cyt) c(6) are crucial to allow subsequent efficient electron transfer to the photooxidized primary donor. To characterize the surface regions of PSI needed for the correct binding of both donors, loop j of PsaB of Chlamydomonas reinhardtii was modified using site-directed mutagenesis and chloroplast transformation. Mutant strains D624K, E613K/D624K, E613K/W627F, and D624K/W627F accumulated <20% of PSI as compared with wild type and were only able to grow photoautotrophically at low light intensities. Mutant strains E613N, E613K, and W627F accumulated >50% of PSI as compared with wild type. This was sufficient to isolate the altered PSI and perform a detailed analysis of the electron transfer between the modified PSI and the two algal donors using flash-induced spectroscopy. Such an analysis indicated that residue Glu(613) of PsaB has two functions: (i) it is crucial for an improved unbinding of the two donors from PSI, and (ii) it orientates the positively charged N-terminal domain of PsaF in a way that allows efficient binding of pc or cyt c(6) to PSI. Mutation of Trp(627) to Phe completely abolishes the formation of an intermolecular electron transfer complex between pc and PSI and also drastically diminishes the rate of electron transfer between the donor and PSI. This mutation also hinders binding and electron transfer between the altered PSI and cyt c(6). It causes a 10-fold increase of the half-time of electron transfer within the intermolecular complex of cyt c(6) and PSI. These data strongly suggest that Trp(627) is a key residue of the recognition site formed by the core of PSI for binding and electron transfer between the two soluble electron donors and the photosystem.