RESUMO
We report simulations of the two-dimensional electronic spectroscopy of the Q(y) band of the D1-D2-Cyt b559 photosystem II reaction center at 77 K. We base the simulations on an existing Hamiltonian that was derived by simultaneous fitting to a wide range of linear spectroscopic measurements and described within modified Redfield theory. The model obtains reasonable agreement with most aspects of the two-dimensional spectra, including the overall peak shapes and excited state absorption features. It does not reproduce the rapid equilibration from high energy to low energy excitonic states evident by a strong cross-peak below the diagonal. We explore modifications to the model to incorporate new structural data and improve agreement with the two-dimensional spectra. We find that strengthening the system-bath coupling and lowering the degree of disorder significantly improves agreement with the cross-peak feature, while lessening agreement with the relative diagonal/antidiagonal width of the 2D spectra. We conclude that two-dimensional electronic spectroscopy provides a sensitive test of excitonic models of the photosystem II reaction center and discuss avenues for further refinement of such models.
Assuntos
Simulação por Computador , Modelos Moleculares , Complexo de Proteína do Fotossistema II/química , Análise Espectral/métodos , Transferência de Energia , Conformação ProteicaRESUMO
Oxidation of H2O by photosystem II is a unique redox reaction in that it requires Ca2+ as well as Cl- as obligatory activators/cofactors of the reaction, which is catalyzed by Mn atoms. The properties of the binding site for Ca2+ in this reaction resemble those of other Ca2+ binding proteins, and recent X-ray structural data confirm that the metal is in fact ligated at least in part by amino acid side chain oxo anions. Removal of Ca2+ blocks water oxidation chemistry at an early stage in the cycle of redox reactions that result in O-O bond formation, and the intimate involvement of Ca2+ in this reaction that is implied by this result is confirmed by an ever-improving set of crystal structures of the cyanobacterial enzyme. Here, we revisit the photosystem II Ca2+ site, in part to discuss the additional information that has appeared since our earlier review of this subject (van Gorkom HJ, Yocum CF In: Wydrzynski TJ, Satoh K (eds) Photosystem II: the light-driven water:plastoquinone oxidoreductase), and also to reexamine earlier data, which lead us to conclude that all S-state transitions require Ca2+.
Assuntos
Cálcio/química , Complexo de Proteína do Fotossistema II/química , Complexo de Proteína do Fotossistema II/metabolismo , Sítios de Ligação , Manganês/química , Oxirredução , Ligação ProteicaRESUMO
Exposure of photosystem II membranes to trypsin that has been treated to inhibit chymotrypsin activity produces limited hydrolysis of manganese stabilizing protein. Exposure to chymotrypsin under the same conditions yields substantial digestion of the protein. Further probing of the unusual insensitivity of manganese stabilizing protein to trypsin hydrolysis reveals that increasing the temperature from 4 to 25 degrees C will cause some acceleration in the rate of proteolysis. However, addition of low (100 microM) concentrations of NH2OH, that are sufficient to reduce, but not destroy, the photosystem II Mn cluster, causes a change in PS II-bound manganese stabilizing protein that causes it to be rapidly digested by trypsin. Immunoblot analyses with polyclonal antibodies directed against the N-terminus of the protein, or against the entire sequence show that trypsin cleavage produces two distinct peptide fragments estimated to be in the 17-20 kDa range, consistent with proposals that there are 2 mol of the protein/mol photosystem II. The correlation of trypsin sensitivity with Mn redox state(s) in photosystem II suggest that manganese stabilizing protein may interact either directly with Mn, or alternatively, that the polypeptide is bound to another protein of the photosystem II reaction center that is intimately involved in binding and redox activity of Mn.
Assuntos
Manganês/química , Complexo de Proteínas do Centro de Reação Fotossintética/química , Complexo de Proteína do Fotossistema II , Proteínas/química , Hidróxido de Amônia , Quimotripsina , Eletroforese em Gel de Poliacrilamida , Hidróxidos , Immunoblotting , Oxirredução , Oxigênio/química , Conformação Proteica , Temperatura , TripsinaRESUMO
PURPOSE: The purpose of this study was to determine the interrater reliability of the active knee-extension test (AKET) using a stabilizing apparatus to measure hamstring length. METHODS: One hundred one subjects (53 girls, 48 boys) ranging in age from 10 to 13 years with no known neuromuscular problems participated. The AKET was performed with subjects lying supine with the hip flexed to 90 degrees with a stabilization device attached to a plinth. Next, subjects were instructed to actively extend the knee until the rater detected myoclonus. Then, the rater flexed the knee until myoclonus subsided and the knee angle was measured with a blinded goniometer. This procedure was repeated by each of three raters. RESULTS: Data were analyzed using ICC (2,1) demonstrating good interrater reliability of 0.79. CONCLUSION: Our results suggest that the AKET, when used with the stabilizing apparatus, demonstrates good interrater reliability for children aged 10 to 13 years.
RESUMO
Photosystem II catalyzes photosynthetic water oxidation. The oxidation of water to molecular oxygen requires four sequential oxidations; the sequentially oxidized forms of the catalytic site are called the S states. An extrinsic subunit, the manganese-stabilizing protein (MSP), promotes the efficient turnover of the S states. MSP can be removed and rebound to the reaction center; removal and reconstitution is associated with a decrease in and then a restoration of enzymatic activity. We have isotopically edited MSP by uniform (13)C labeling of the Escherichia coli-expressed protein and have obtained the Fourier transform infrared spectrum associated with the S(1) to S(2) transition in the presence either of reconstituted (12)C or (13)C MSP. (13)C labeling of MSP is shown to cause 30-60 cm(-1) shifts in a subset of vibrational lines. The derived, isotope-edited vibrational spectrum is consistent with a deprotonation of glutamic/aspartic acid residues on MSP during the S(1) to S(2) transition; the base, which accepts this proton(s), is not located on MSP. This finding suggests that this subunit plays a role as a stabilizer of a charged transition state and, perhaps, as a general acid/base catalyst of oxygen evolution. These results provide a molecular explanation for known MSP effects on oxygen evolution.
Assuntos
Manganês/metabolismo , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Complexo de Proteína do Fotossistema II , Proteínas/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Escherichia coli , Peso Molecular , Oxirredução , Fotoquímica , Conformação Proteica , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
In solution, Manganese Stabilizing Protein, the polypeptide which is responsible for the structural and functional integrity of the manganese cluster in photosystem II, is a natively unfolded protein with a prolate ellipsoid shape [Lydakis-Simantiris et al. (1999) Biochemistry 38, 404-414; Zubrzycki et al. (1998) Biochemistry 37, 13553-13558]. The C-terminal tripeptide of Manganese Stabilizing Protein was shown to be critical for binding to photosystem II and restoration of O(2) evolution activity [Betts et al. (1998) Biochemistry 37, 14230-14236]. Here, we report new biochemical, hydrodynamic, and spectroscopic data on mutants E246K, E246STOP, L245E, L245STOP, and Q244STOP. Truncation of the final dipeptide (E246STOP) or substitution of Glu246 with Lys resulted in no significant changes in secondary and tertiary structures of Manganese Stabilizing Protein as monitored by CD spectroscopy. The apparent molecular mass of the protein remained unchanged, both mutants were able to rebind to photosystem II, and both proteins reactivate O(2) evolution. Manganese Stabilizing Protein lacking the final tripeptide (L245STOP), or substitution of Glu for Leu245 dramatically modified the protein's solution structure. The apparent molecular masses of these mutants increased significantly, which might indicate unfolding of the protein in solution. This was verified by CD spectroscopy. Both mutant proteins rebound to photosystem II with lower affinities, and activation of O(2) evolution was decreased dramatically. Enhancement of these defects was observed upon removal of the final tetrapeptide (Q244STOP). These results indicate that Leu245 is essential to maintaining Manganese Stabilizing Protein's solution structure in a conformation that promotes efficient binding to photosystem II and/or for the subsequent steps that lead to enzyme activation. Based on an analysis of the properties of C-terminal mutations, a hypothesis for structural requirements for functional binding of Manganese Stabilizing Protein to photosystem II is presented. Effects of C-terminal mutations on the UV spectrum of Manganese Stabilizing Protein were also examined. Mutations that alter solution structure also affect a 293 nm absorption shoulder which is assigned to the only tryptophan residue, Trp241, in the protein, and this absorbance feature is shown to be a useful indicator of alterations to the Trp241 environment.
Assuntos
Leucina/química , Manganês/química , Metaloproteínas/química , Complexo de Proteínas do Centro de Reação Fotossintética/química , Complexo de Proteína do Fotossistema II , Dobramento de Proteína , Proteínas/química , Dicroísmo Circular , Leucina/genética , Metaloproteínas/genética , Mutagênese Sítio-Dirigida , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Estrutura Secundária de Proteína/genética , Proteínas/genética , Deleção de Sequência , Spinacia oleracea , Triptofano/genéticaRESUMO
Developments in manganese biochemistry have centered on the discovery of new manganese enzymes, X-ray analysis of binuclear manganese enzymes, and the discovery of new spectroscopic signatures for the oxygen-evolving complex. Despite these gains, many questions regarding the structure, composition and redox state of the oxygen-evolving complex remain unanswered.
Assuntos
Manganês/metabolismo , Arginase/química , Arginase/metabolismo , Catalase/química , Catalase/metabolismo , Oxirredução , Água/químicaRESUMO
Photosystem II, the multisubunit protein complex that oxidizes water to O2, requires the inorganic cofactors Ca2+ and Cl- to exhibit optimal activity. Chloride can be replaced functionally by a small number of anionic cofactors (Br-, NO3-, NO2-, I-), but among these anions, only Br- is capable of restoring rates of oxygen evolution comparable to those observed with Cl-. UV absorption difference spectroscopy was utilized in the experiments described here as a probe to monitor donor side reactions in photosystem II in the presence of Cl- or surrogate anions. The rate of the final step of the water oxidation cycle was found to depend on the activating anion bound at the Cl- site, but the kinetics of this step did not limit the light-saturated rate of oxygen evolution. Instead, the lower oxygen evolution rates supported by surrogate anions appeared to be correlated with an instability of the higher oxidation states of the oxygen-evolving complex that was induced by addition of these anions. Reduction of these states takes place not only with I- but also with NO2- and to a lesser extent even with NO3- and Br- and is not related to the ability of these anions to bind at the Cl- binding site. Rather, it appears that these anions can attack higher oxidation states of the oxygen evolving complex from a second site that is not shielded by the extrinsic 17 and 23 kDa polypeptides and cause a one-electron reduction. The decrease of the oxygen evolution rate may result from accumulated damage to the reaction center protein by the one-electron oxidation product of the anion.
Assuntos
Ânions/química , Oxigênio/química , Complexo de Proteínas do Centro de Reação Fotossintética/química , Sítios de Ligação , Cloretos/química , Transporte de Elétrons , Cinética , Oxirredução , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Complexo de Proteína do Fotossistema II , Espectrofotometria Ultravioleta , Spinacia oleracea , Água/químicaRESUMO
The thermostability of manganese stabilizing protein of photosystem II was examined by biochemical and spectroscopic techniques. Samples of both native and recombinant spinach manganese stabilizing protein incubated at 90 degreesC and then cooled to 25 degreesC were capable of rebinding to, and of reactivating, the O2-evolution activity of photosystem II membranes from which the native protein had been removed. Far-UV circular dichroism and FT-IR spectroscopies were used to analyze the structural consequences of heating manganese stabilizing protein. The data obtained from these techniques show that heating causes a complete loss of the protein's secondary structure, and that this is a reversible, noncooperative phenomenon. Upon cooling, the secondary structures of the heat-treated proteins return to a state similar to, but not identical with, that of the native, unheated controls. Restoration of a near-native tertiary structure is confirmed both by size-exclusion chromatography and by near-UV circular dichroism. The functional and structural thermostability of manganese stabilizing protein reported here, in conjunction with additional known properties of this protein (acidic pI, high random coil and turn content, anomalous hydrodynamic behavior), identifies manganese stabilizing protein as a natively unfolded protein [Weinreb et al. (1996) Biochemistry 35, 13709-13715]. Although these proteins lack amino acid sequence identity, their functional solution conformations under physiological conditions are said to be "natively unfolded". We suggest that, as with other members of this family of proteins, the natively unfolded structure of manganese stabilizing protein facilitates the highly effective protein-protein interactions that are necessary for its assembly into photosystem II.
Assuntos
Manganês/química , Peptídeos/química , Complexo de Proteínas do Centro de Reação Fotossintética/química , Complexo de Proteína do Fotossistema II , Dobramento de Proteína , Proteínas/química , Cromatografia em Gel , Dicroísmo Circular , Temperatura Alta , Manganês/metabolismo , Oxigênio/metabolismo , Peptídeos/metabolismo , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Desnaturação Proteica , Estrutura Secundária de Proteína , Proteínas/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier , Spinacia oleraceaRESUMO
The extrinsic manganese stabilizing protein of photosystem II is required for Mn retention by the O2-evolving complex, accelerates the rate of O2 evolution, and protects photosytem II against photoinhibition. We report results from studies of the in vitro reconstitution of spinach photosytem II with recombinant manganese stabilizing protein with C-terminal deletions of two, three, and four amino acids. The deletions were the result of amber mutations introduced by site-directed mutagenesis. Removal of the C-terminal dipeptide (Glu-Gln) did not diminish the ability of the manganese stabilizing protein either to rebind to or to restore high rates of O2 evolution to photosystem II preparations depleted of the native protein. Deletion of the C-terminal tripeptide (Leu-Glu-Gln) resulted in weakened but specific binding of manganese stabilizing protein to photosystem II and minimal recovery of O2 evolution activity. Removal of the C-terminal tetrapeptide (Gln-Leu-Glu-Gln) eliminated the ability of the subunit to interact stably with all of its available binding sites on photosystem II, as evidenced by the fact that this mutant was totally inactive in restoring O2 evolution activity. Evidence is presented to indicate that these mutational effects on the binding and function of the manganese stabilizing protein may be due to major changes in tertiary structure. The truncation mutations lacking either the C-terminal tri- or tetrapeptide exhibit apparent size increases of 25 and 40%, respectively, when compared either to a mutant lacking the C-terminal dipeptide or to the wild-type protein.
Assuntos
Manganês/metabolismo , Oxigênio/metabolismo , Fragmentos de Peptídeos/fisiologia , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Complexo de Proteína do Fotossistema II , Proteínas/fisiologia , Arabidopsis , Cromatografia Líquida de Alta Pressão , Escherichia coli/genética , Glutamina/genética , Leucina/genética , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Proteínas/genética , Proteínas/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Spinacia oleraceaRESUMO
The Cl- binding properties in the successive oxidation states of the O2 evolving complex of photosystem II were investigated by measurements of UV absorbance changes, induced by a series of saturating flashes, that monitor manganese oxidation state transitions. In dark-adapted, intact photosystem II, Cl- can be replaced by NO3- in minutes, in an exchange reaction that depends on the NO3- concentration and that is not rate-limited by dissociation of Cl- from its binding site. Preillumination of dark-adapted photosystem II by one or two flashes accelerated the NO3- substitution reaction by an order of magnitude. A quantitative analysis of the Cl- concentration dependence of UV absorbance changes, measured in photosystem II preparations depleted of extrinsic 17 and 23 kDa polypeptides, shows that the Cl- binding properties of photosystem II change with the oxidation state of the oxygen evolving complex. Although the affinity for the individual S-states could not be determined with precision, it is shown that the affinity is an order of magnitude lower in the S2 state than in the S1 state. Comparison of the results obtained using intact photosystem II and preparations depleted of the 17 and 23 kDa extrinsic polypeptides suggests that these proteins constitute a diffusion barrier, which prevents fast equilibration of the Cl- binding site with the medium, but does not change the Cl- affinity of the binding site.
Assuntos
Cloretos/metabolismo , Oxigênio/metabolismo , Peptídeos/metabolismo , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Sítios de Ligação , Luz , Fotoquímica , Complexo de Proteína do Fotossistema II , Spinacia oleracea , TermodinâmicaRESUMO
Photosystem II catalyzes the light-driven oxidation of water and reduction of plastoquinone in oxygenic photosynthesis. The manganese stabilizing protein (MSP) of photosystem II is an extrinsic subunit that plays an important role in catalytic activity. This subunit can be extracted and re-bound to the photosystem II reaction center. Extraction is associated with decreased stability of manganese binding by the enzyme and by loss in high rates of oxygen evolution activity; reconstitution reverses these phenomena. Since little is known about the assembly of complex membrane proteins, we have employed isotope editing and vibrational spectroscopy to obtain information about any changes in secondary structure that occur in MSP upon functional reconstitution to photosystem II. The spectroscopic data obtained are consistent with substantial changes in conformation when MSP binds to photosystem II; approximately 30-40% of the peptide backbone undergoes a change in secondary structure. These conclusions were reached by comparing different aliquots, before and after binding, of the same 13[C]MSP sample. Analysis of amide I band line shapes through Fourier deconvolution and nonlinear regression suggests that binding of MSP to photosystem II is associated with a decrease in random structure and an increase in beta-sheet content. We conclude that binding of MSP to the reaction center can induce folding of MSP. Our results also indicate that, in solution, MSP can sample a variety of conformational states, which differ in hydrogen bonding of the peptide backbone.
Assuntos
Complexo de Proteínas do Centro de Reação Fotossintética/química , Complexo de Proteína do Fotossistema II , Conformação Proteica , Proteínas/química , Western Blotting , Carbono , Isótopos de Carbono , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Oxigênio/metabolismo , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Ligação Proteica , Biossíntese de Proteínas , Estrutura Secundária de Proteína , Proteínas/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Espectroscopia de Infravermelho com Transformada de Fourier , Spinacia oleraceaRESUMO
The 33-kDa protein of the photosynthetic O2-evolving complex, also known as manganese stabilizing protein, contributes to the structural stability of the photosystem II tetranuclear Mn cluster and stimulates the water-oxidizing activity of this cluster. Quantification of extrinsic polypeptides in photosystem II has yielded data that support stoichiometries of either one or two copies of each protein per photosystem II reaction center. We recently described the cold-sensitive assembly of a mutant 33-kDa protein with a single amino acid replacement (Val235Ala) [Betts, S. D., Ross, J. R., Pichersky, E., & Yocum, C. F. (1996) Biochemistry 35, 6302-6307]. We have extended the characterization of this mutation. When photosystem II membranes depleted of the 33 kDa extrinsic protein are exposed to mixtures of wild type and Val235Ala manganese stabilizing protein, binding of the wild type protein is strongly preferred. If, however, protein containing the Val235Ala mutation is first bound to photosystem II only half of this protein (about 1 mol/mol of photosystem II reaction centers) is susceptible to displacement by the wild type protein, even after multiple exposures to the latter. These results support the conclusion that 2 mol of manganese stabilizing protein are bound per reaction center. Our data show as well that the mutant 33-kDa protein competes with the wild type protein for at least one of two binding sites on photosystem II and that the mutant protein binds tightly to only one of two sites. These results demonstrate that the two binding sites on photosystem II for the 33-kDa protein have different properties with respect to recognition and binding of this protein.
Assuntos
Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Complexo de Proteína do Fotossistema II , Proteínas/genética , Arabidopsis/metabolismo , Sítios de Ligação , Ligação Competitiva , Eletroforese em Gel de Poliacrilamida , Mutação , Ligação Proteica , Proteínas/química , Spinacia oleracea/metabolismo , TemperaturaRESUMO
The Cl- requirement in the redox cycle of the oxygen-evolving complex (OEC) was determined by measurements of flash-induced UV absorbance changes in Cl(-)-depleted and Cl(-)-reconstituted photosystem II membranes. On the first flash after dark adaptation the spectrum and amplitude of those changes, known to reflect the oxidation of MnIII to MnIV on the S1-->S2 transition, were the same in the presence or absence of Cl-. On the second and later flashes, however, absorbance changes in Cl(-)-depleted samples revealed only electron transfer from tyrosine to quinone which reversed slowly in the dark by charge recombination and did not produce the S3-state. A rapid method was developed to remove Cl- after producing the S3-state by two flashes. The lifetime of the S3-state was found to be unaffected by Cl(-)-depletion, in contrast to the 20-fold stabilization of the S2 lifetime by Cl- removal, and the Cl(-)-depleted S3-state did not proceed to S0 on flash illumination. However, when the same Cl(-)-depletion procedure was applied after producing the S0-state by three flashes, further advance to S2 by two additional flashes was not impaired by the absence of Cl-. The requirement for Cl- only on the S2-->S3 and S3-->S0 transitions can be rationalized by the hypothesis that Cl- is required for electron transfer between manganese ions within the oxygen-evolving complex.
Assuntos
Cloretos/metabolismo , Oxirredutases/metabolismo , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Oxirredução , Complexo de Proteína do Fotossistema II , Espectrofotometria Atômica , Espectrofotometria UltravioletaRESUMO
The 33-kDa extrinsic subunit of PSII stabilizes the O2-evolving tetranuclear Mn cluster and accelerates O2 evolution. We have used site-directed mutagenesis to replace one or both Cys residues in spinach MSP with Ala. Previous experiments using native and reduced MSP led to the conclusion that a disulfide bridge between these two cysteines is essential both for its binding and its functional properties. We report here that the disulfide bridge, though essential for MSP stability, is otherwise dispensible. The mutation C51A by itself had a delayed effect on MSP function: [C51A]MSP restored normal rates of O2 evolution to PSII but was defective in stabilizing this activity during extended illumination. In contrast, the Cys-free double mutant, [C28A,C51A]MSP, was functionally identical to the wild-type protein. Based on results presented here, we propose a light-dependent interaction between MSP and PSII that occurs only during the redox cycling of the Mn cluster and which is destabilized by the single mutation, C51A.
Assuntos
Dissulfetos/química , Manganês/farmacologia , Mutagênese Sítio-Dirigida , Complexo de Proteínas do Centro de Reação Fotossintética/química , Oxigênio/metabolismo , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Complexo de Proteína do Fotossistema II , Conformação Proteica , Proteínas Recombinantes , Spinacia oleracea , Relação Estrutura-AtividadeRESUMO
Photosystem II (PSII) is a multisubunit transmembrane protein complex that oxidizes water and evolves O2. A tetranuclear manganese cluster associated with integral membrane subunits of PSII catalyzes water oxidation. The 33-kDa water-soluble PSII subunit, or manganese-stabilizing protein (MSP), stabilizes the O2-evolving manganese cluster and accelerates O2 evolution. Spinach PSII can be depleted of native MSP under conditions which retain a functional manganese cluster. Reconstition of MSP-depleted PSII with recombinant MSP was equally efficient at 4 and 22 degrees C. Replacement of Val235 (a conserved residue near the C-terminus of MSP) with Ala inhibited assembly of MSP at 4 degrees C, but not at 22 degrees C. Once assembled, [V235A]MSP remained bound to PSII even at 4 degrees C and in the presence of low concentrations of urea. Results from far-UV circular dichroism spectrometry indicated that [V235A]-MSP was destabilized by low temperature to a greater extent than the wild-type protein. However, the effect of temperature on the secondary structure of both the mutant and wild-type proteins was small compared to the temperature-independent destabilization of secondary structure induced by the mutation. These results demonstrate that the V235A mutation introduces an activation energy barrier for assembly of MSP into PSII, and it is suggested that the mutation acts by inhibiting isomerization of one or more prolyl peptide bonds required for assembly.
Assuntos
Complexo de Proteínas do Centro de Reação Fotossintética/química , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Complexo de Proteína do Fotossistema II , Mutação Puntual , Proteínas , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Temperatura Baixa , Substâncias Macromoleculares , Manganês/química , Manganês/metabolismo , Metaloproteínas/química , Metaloproteínas/genética , Metaloproteínas/metabolismo , Dados de Sequência Molecular , Estrutura Molecular , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Dobramento de Proteína , Homologia de Sequência de Aminoácidos , Spinacia oleracea/genética , Spinacia oleracea/metabolismo , TermodinâmicaRESUMO
An intrinsic 22 kDa polypeptide is associated with the O2-evolving Photosystem II core complex in a variety of green plants, although it does not appear to be required for O2 evolution. Digestion of thylakoid membranes and isolated Photosystem II preparations with trypsin, followed by immunoblotting using spinach anti-22 kDa antibodies, leads to two observations: (1) the domain between the 2nd and 3rd transmembrane helices of the 22 kDa protein is stromally exposed, and (2) only in a reaction center complex preparation, lacking the chlorophyll a/b-light harvesting complex II, is there extensive proteolytic cleavage of the 22 kDa protein. We also found that after, but not prior to, selective extraction of the 22 and 10 kDa proteins from Photosystem II membranes, the chlorophyll a/b-light harvesting complex II can be separated from the Photosystem II reaction center core by precipitation with MgCl2. This result suggests that the 22 kDa polypeptide is located between the Photosystem II reaction center polypeptides and light-harvesting complex II; it is possible that the protein serves as a link between the two protein complexes. The presence of the 22 kDa protein in several species was also examined by immunoblotting with polyclonal spinach anti-22 kDa antibodies.
Assuntos
Complexo de Proteínas do Centro de Reação Fotossintética/química , Spinacia oleracea/química , Sequência de Aminoácidos , Complexos de Proteínas Captadores de Luz , Dados de Sequência Molecular , Peptídeos/química , Complexo de Proteína do Fotossistema IIRESUMO
The psbO gene of cyanobacteria, green algae and higher plants encodes the precursor of the 33 kDa manganese-stabilizing protein (MSP), a water-soluble subunit of photosystem II (PSII). Using a pET-T7 cloning/expression system, we have expressed in Escherichia coli a full-length cDNA clone of psbO from Arabidopsis thaliana. Upon induction, high levels of the precursor protein accumulated in cells grown with vigorous aeration. In cells grown under weak aeration, the mature protein accumulated upon induction. In cells grown with moderate aeration, the ratio of precursor to mature MSP decreased as the optical density at induction increased. Both forms of the protein accumulated as inclusion bodies from which the mature protein could be released under mildly denaturing conditions that did not release the precursor. Renatured Arabidopsis MSP was 87% as effective as isolated spinach MSP in restoring O2 evolution activity to MSP-depleted PSII membranes from spinach; however, the heterologous protein binds to spinach PSIIs with about half the affinity of the native protein. We also report a correction to the previously published DNA sequence of Arabidopsis psbO (Ko et al., Plant Mol Biol 14 (1990) 217-227).
Assuntos
Arabidopsis/química , Proteínas de Bactérias/metabolismo , Metaloproteínas/metabolismo , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Complexo de Proteína do Fotossistema II , Proteínas , Proteínas Recombinantes/metabolismo , Spinacia oleracea/química , Sequência de Aminoácidos , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , DNA Complementar , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Corpos de Inclusão/metabolismo , Metaloproteínas/isolamento & purificação , Dados de Sequência Molecular , Oxigênio/metabolismo , Complexo de Proteínas do Centro de Reação Fotossintética/biossíntese , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Complexo de Proteínas do Centro de Reação Fotossintética/isolamento & purificação , Precursores de Proteínas/biossíntese , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Análise de Sequência de DNARESUMO
We report the fluorescence decay kinetics and the vibrational properties of chlorophyll a bound to the 47-kDa antenna protein (CP47) of spinach photosystem II. The chlorophyll fluorescence of CP47 samples decays with four lifetimes (tau = 75.8 ps, 1.05 ns, 3.22 ns, and 5.41 ns). The 75.8-ps and 3.22-ns components are associated with chlorophyll a bound to relatively intact centers, the 1.05-ns component corresponds to chlorophyll bound to centers that are slightly perturbed, and the the 5.41-ns phase probably originates from centers that are severely denatured. The resonance Raman spectrum of CP47 at 441.6 nm (this work) and at 406.7 nm [de Paula, J. C., Ghanotakis, D. F., Bowlby, N. R., Dekker, J. P., Yocum, C. F., & Babcock, G. T. (1990) in Current Research in Photosynthesis (Baltscheffsky, M., Ed.), Vol. I, pp 643-646, Kluwer Academic Publishers, Dordrecht, The Netherlands] shows heterogeneity in the C = O stretching region. This part of the spectrum monitors the environment of the keto group at position 9 of the chlorophyll a molecule. We show that several structurally distinct pools of chlorophyll a are bound to CP47. Four of these may be distinguished by their C9 = O stretching frequencies (nu C = O = 1670, 1688, 1693, and 1701 cm-1). By analyzing the resonance enhancement pattern of these modes, we ascribe the 1693-cm-1 vibration to denatured centers. Of the remaining populations, we propose that the 1670-cm-1 vibration is consistent with a hydrogen bond between the C9 = O group of chlorophyll a and the protein. We elaborate on the role of this chromophore-protein interaction in the mechanism of energy transfer within the 47-kDa antenna protein.