Biomedical subjects

Naoki Mizusawa

Publications and source records attributed to Naoki Mizusawa.

8 recordsLinked to original sources

PubMed2005

FTIR detection of structural changes in a histidine ligand during S-state cycling of photosynthetic oxygen-evolving complex.

Changes in structural coupling between the Mn cluster and a putative histidine ligand during the S-state cycling of the oxygen-evolving complex (OEC) have been detected directly by Fourier transform infrared (FTIR) spectroscopy in photosystem (PS) II core particles from the cyanobacterium Synechocystis sp. PCC6803, in which histidine residues were selectively labeled with l-[(15)N(3)]histidine. The bands sensitive to the histidine-specific isotope labeling appeared at 1120-1090 cm(-)(1) in the spectra induced upon the first-, second-, and fourth-flash illumination, for the S(2)/S(1), S(3)/S(2), and S(1)/S(0) differences, at similar frequencies with different sign and/or intensity depending on the respective S-state transitions. However, no distinctive band was observed in the third-flash induced spectrum for the S(0)/S(3) difference. The results indicate that a single histidine residue coupled with the structural changes of the OEC during the S-state cycling is responsible for the observed histidine bands, in which the histidine modes changed during the S(0)-to-S(1) transition are reversed upon the S(1)-to-S(2) and S(2)-to-S(3) transitions. The 1186(+)/1178(-) cm(-)(1) bands affected by l-[(15)N(3)]histidine labeling were observed only for the S(2)/S(1) difference, but those affected by universal (15)N labeling appeared prominently showing a clear S-state dependency. Possible origins of these bands and changes in the histidine modes during the S-state cycling are discussed.

Bacterial Proteins↗

PubMed2005

Changes in structural and functional properties of oxygen-evolving complex induced by replacement of D1-glutamate 189 with glutamine in photosystem II: ligation of glutamate 189 carboxylate to the manganese cluster.

A carboxylate group of D1-Glu-189 in photosystem II has been proposed to serve as a direct ligand for the manganese cluster. Here we constructed a mutant that eliminates the carboxylate by replacing D1-Glu-189 with Gln in the cyanobacterium Synechocystis sp. PCC 6803, and we examined the resulting effects on the structural and functional properties of the oxygen-evolving complex (OEC) in photosystem II. The E189Q mutant grew photoautotrophically, and isolated photosystem II core particles evolved oxygen at approximately 70% of the rate of control wild-type particles. The E189Q OEC showed typical S(2) state electron spin resonance signals, and the spin center distance between the S(2) state manganese cluster and the Y(D) (D2-Tyr-160), detected by electron-electron double resonance spectroscopy, was not affected by this mutation. However, the redox potential of the E189Q OEC was considerably lower than that of the control OEC, as revealed by the elevated peak temperature of the S(2) state thermoluminescence bands. The mutation resulted in specific changes to bands ascribed to the putative carboxylate ligands for the manganese cluster and to a few carbonyl bands in mid-frequency (1800 to 1100 cm(-1)) S(2)/S(1) Fourier transform infrared difference spectrum. Notably, the low frequency (650 to 350 cm(-1)) S(2)/S(1) Fourier transform infrared difference spectrum was also uniquely changed by this mutation in the frequencies for the manganese cluster core vibrations. These results suggested that the carboxylate group of D1-Glu-189 ligates the manganese ion, which is influenced by the redox change of the oxidizable manganese ion upon the S(1) to S(2) transition.

Amino Acid Sequence↗

PubMed2004

Changes in the functional and structural properties of the Mn cluster induced by replacing the side group of the C-terminus of the D1 protein of photosystem II.

A free alpha-COO(-) in the C-terminal alanine-344 (Ala344) in the D1 protein of photosystem II is thought to be responsible for ligating the Mn cluster. The effects of the side group of the C-terminus of the D1 protein on the functional and structural properties of the oxygen-evolving complex (OEC) were comprehensively studied by replacing Ala344 with glycine (Gly), valine (Val), aspartate (Asp), or asparagine (Asn). All the mutants grew photoautotrophically under low-light conditions with lower O(2) evolution activity depending on the mutants when compared with the activity of the control wild type. The Gly-, Asp-, and Asn-substituted mutants did not grow under high-light conditions, while the Val-substituted mutant grew even under the high-light conditions. S(2)-state thermoluminescence bands appeared at slightly elevated temperatures when compared with those of the wild type in the Asp- and Gly-substituted mutants, but at almost normal temperatures in the Val- and Asn-substituted mutants. The oxygen-evolving core particles isolated from the mutants showed little change in protein composition. The Gly-, Asp-, and Asn-substituted core particles exhibited low-temperature electron spin resonance (ESR) spectra with reduced S(2) multiline and enhanced g = 4.1 ESR signals, while the Val-substituted particles showed a spectrum similar to that of the control particles. Mid-frequency Fourier transform infrared difference spectra showed distinctive changes in several bands arising from the putative carboxylate ligands for the Mn cluster in all substituted particles, but the bands for the putative C-terminal alpha-carboxylate did not seem to change in the substituted spectra. The changes induced by the Asp and Asn substitution resembled each other except for the amide I region, and showed some similarity to those induced by the Gly substitution in the symmetric carboxylate stretching region. The results were interpreted to mean that similar types of changes of the carboxylate ligands are induced by these substitutions. The band from a putative histidine ligand for the Mn cluster was similarly affected in the Gly-, Asp-, and Asn-substituted spectra, but not in the Val-substituted spectrum. Notably, marked changes in the amide I, amide II, and carboxylate bands were observed in the Val-substituted spectrum, which was different from the Gly-, Asp-, and Asn-substituted spectra. The results indicated that the structural perturbations induced by the Val substitution include large changes of the protein backbone and are considerably different from those induced by the other substitutions. Possible amino acid ligands participating in the changes deduced by Ala344 replacement in the D1 C-terminal and the effects of the changes of the side group on these ligands were considered on the basis of the available X-ray model of the OEC.

Amino Acid Substitution↗

PubMed2004

Structural changes of D1 C-terminal alpha-carboxylate during S-state cycling in photosynthetic oxygen evolution.

Changes in the chemical structure of alpha-carboxylate of the D1 C-terminal Ala-344 during S-state cycling of photosynthetic oxygen-evolving complex were selectively measured using light-induced Fourier transform infrared (FTIR) difference spectroscopy in combination with specific [(13)C]alanine labeling and site-directed mutagenesis in photosystem II core particles from Synechocystis sp. PCC 6803. Several bands for carboxylate symmetric stretching modes in an S(2)/S(1) FTIR difference spectrum were affected by selective (13)C labeling of the alpha-carboxylate of Ala with l-[1-(13)C]alanine, whereas most of the isotopic effects failed to be induced in a site-directed mutant in which Ala-344 was replaced with Gly. Labeling of the alpha-methyl of Ala with l-[3-(13)C]alanine had much smaller effects on the spectrum to induce isotopic bands due to a symmetric CH(3) deformation coupled with the alpha-carboxylate. The isotopic bands for the alpha-carboxylate of Ala-344 showed characteristic changes during S-state cycling. The bands appeared prominently upon the S(1)-to-S(2) transition and to a lesser extent upon the S(2)-to-S(3) transition but reappeared at slightly upshifted frequencies with the opposite sign upon the S(3)-to-S(0) transition. No obvious isotopic band appeared upon the S(0)-to-S(1) transition. These results indicate that the alpha-carboxylate of C-terminal Ala-344 is structurally associated with a manganese ion that becomes oxidized upon the S(1)-to-S(2) transition and reduced reversely upon the S(3)-to-S(0) transition but is not associated with manganese ion(s) oxidized during the S(0)-to-S(1) (and S(2)-to-S(3)) transition(s). Consistently, l-[1-(13)C]alanine labeling also induced spectral changes in the low frequency (670-350 cm(-1)) S(2)/S(1) FTIR difference spectrum.

Alanine↗

PubMed2004

Mid- to low-frequency Fourier transform infrared spectra of S-state cycle for photosynthetic water oxidation in Synechocystis sp. PCC 6803.

Flash-induced Fourier transform infrared (FTIR) difference spectra for the four-step S-state cycle and the effects of global (15)N- and (13)C-isotope labeling on the difference spectra were examined for the first time in the mid- to low-frequency (1200-800 cm(-1)) as well as the mid-frequency (1700-1200 cm(-1)) regions using photosystem (PS) II core particles from cyanobacterium Synechocystis sp. PCC 6803. The difference spectra clearly exhibited the characteristic vibrational features for each transition during the S-state cycling. It is likely that the bands that change their sign and intensity with the S-state advances reflect the changes of the amino acid residues and protein matrices that have functional and/or structural roles within the oxygen-evolving complex (OEC). Except for some minor differences, the trends of S-state dependence in the 1700-1200 cm(-1) frequency spectra of the PS II cores from Synechocystis were comparable to that of spinach, indicating that the structural changes of the polypeptide backbones and amino acid side chains that occur during the oxygen evolution are inherently identical between cyanobacteria and higher plants. Upon (13)C-labeling, most of the bands, including amide I and II modes and carboxylate stretching modes, showed downward shifts; in contrast, (15)N-labeling induced isotopic shifts that were predominantly observed in the amide II region. In the mid- to low-frequency region, several bands in the 1200-1140 cm(-1) region were attributable to the nitrogen- and/or carbon-containing group(s) that are closely related to the oxygen evolution process. Specifically, the putative histidine ligand exhibited a band at 1113 cm(-1) which was affected by both (15)N- and (13)C-labeling and showed distinct S-state dependency. The light-induced bands in the 900-800 cm(-1) region were downshifted only by (13)C-labeling, whereas the bands in the 1000-900 cm(-1) region were affected by both (15)N- and (13)C-labeling. Several modes in the mid- to low-frequency spectra were induced by the change in protonation state of the buffer molecules accompanied by S-state transitions. Our studies on the light-induced spectrum showed that contributions from the redox changes of Q(A) and the non-heme iron at the acceptor side and Y(D) were minimal. It was, therefore, suggested that the observed bands in the 1000-800 cm(-1) region include the modes of the amino acid side chains that are coupled to the oxidation of the Mn cluster. S-state-dependent changes were observed in some of the bands.

Cyanobacteria↗

PubMed2004

Impact of replacement of D1 C-terminal alanine with glycine on structure and function of photosynthetic oxygen-evolving complex.

The C-terminal alanine 344 (Ala-344) in the D1 protein of photosystem II is conserved in all of the organisms performing oxygenic photosynthesis. A free alpha-COO(-) of Ala-344 has been proposed to be responsible for ligating the Mn cluster. Here, we constructed a mutant having D1 in which D1-Ala-344 was replaced with glycine (Gly) in cyanobacterium Synechocystis sp. PCC 6803. The effects of this minimal change in the side group from methyl to hydrogen on the properties of the oxygen-evolving complex were comprehensively investigated using purified core particles. The mutant grew photoautotrophically, and little change was observed in the protein composition of the oxygen-evolving core particles. The Gly-substituted oxygen-evolving complex showed small but normal S(2) multiline and enhanced g = 4.1 electron spin resonance signals and S(2)-state thermoluminescence bands with slightly elevated peak temperature. The Gly substitution resulted in distinct but relatively small changes in a few bands arising from the putative carboxylate ligand for the Mn cluster in the mid-frequency (1800-1000 cm(-1)) S(2)/S(1) Fourier transform infrared difference spectrum. In contrast, the low frequency (670-350 cm(-1)) S(2)/S(1) Fourier transform infrared difference spectrum was markedly changed by the substitution. The results indicate that the internal structure of the Mn cluster and/or the interaction between the Mn cluster and its ligand are considerably altered by a simple change in the side group, from methyl to hydrogen, at the C-terminal of the D1 protein.

Alanine↗

PubMed2003

Changes of low-frequency vibrational modes induced by universal 15N- and 13C-isotope labeling in S2/S1 FTIR difference spectrum of oxygen-evolving complex.

The effects of universal (15)N- and (13)C-isotope labeling on the low- (650-350 cm(-1)) and mid-frequency (1800-1200 cm(-1)) S(2)/S(1) Fourier transform infrared (FTIR) difference spectrum of the photosynthetic oxygen-evolving complex (OEC) were investigated in histidine-tagged photosystem (PS) II core particles from Synechocystis sp. PCC 6803. In the mid-frequency region, the amide II modes were predominantly affected by (15)N-labeling, whereas, in addition to the amide II, the amide I and carboxylate modes were markedly affected by (13)C-labeling. In the low-frequency region, by comparing a light-induced spectrum in the presence of ferricyanide as the electron acceptor, with the double difference S(2)/S(1) spectrum obtained by subtracting the Q(A)(-)/Q(A) from the S(2)Q(A)(-)/S(1)Q(A) spectrum, considerable numbers of bands found in the light-induced spectrum were assigned to the S(2)/S(1) vibrational modes in the unlabeled PS II core particles. Upon (13)C-labeling, changes were observed for most of the prominent bands in the S(2)/S(1) spectrum. Although (15)N-labeling also induced changes similar to those by (13)C-labeling, the bands at 616(-), 605(+), 561(+), 555(-), and 544(-) cm(-1) were scarcely affected by (15)N-labeling. These results indicated that most of the vibrational modes found in the low-frequency spectrum are derived from the coupling between the Mn-cluster and groups containing nitrogen and/or carbon atom(s) in a direct manner and/or through hydrogen bonding. Interestingly, an intensive band at 577(-) cm(-1) was not affected by (15)N- and (13)C-isotope labeling, indicating that this band arises from the mode that does not include either nitrogen or carbon atoms, such as the skeletal vibration of the Mn-cluster or stretching vibrational modes of the Mn-ligand.

Carbon Isotopes↗

PubMed2003

Degradation of the D1 protein of photosystem II under illumination in vivo: two different pathways involving cleavage or intermolecular cross-linking.

The D1 protein of the photosystem II reaction center turns over the most rapidly of all the proteins of the thylakoid membrane under illumination in vivo. In vitro, the D1 protein sustained cleavage in a surface-exposed loop (DE loop) or cross-linking with another reaction center protein, the D2 protein or cytochrome b(559), under illumination. We found that the D1 protein was damaged in essentially the same way in vivo, although the resultant fragments and cross-linked adducts barely accumulated due to digestion by proteases. In vitro studies detected a novel stromal protease(s) that digested the adducts but not the monomeric D1 protein. These observations suggest that, in addition to cleavage, the cross-linking reactions themselves are processes involved in complete degradation of the D1 protein in vivo. Peptide mapping experiments located the cross-linking sites with the D2 protein among residues 226-244, which includes the cross-linking site with cytochrome b(559) [Barbato, R., et al. (1995) J. Biol. Chem. 270, 24032-24037], in the N-terminal part of the DE loop, while N-terminal amino acid sequencing of the fragment located the cleavage site around residue 260 in the C-terminal part of the loop. We propose a model explaining the occurrence of simultaneous cleavage and cross-linking and discuss the mechanisms of complete degradation of the D1 protein in vivo.

Chloroplasts↗