BBA - Bioenergetics (v.1777, #4)

Compelling EPR evidence that the alternative oxidase is a diiron carboxylate protein by Anthony L. Moore; Jane E. Carré; Charles Affourtit; Mary S. Albury; Paul G. Crichton; Kiyoshi Kita; Peter Heathcote (327-330).
The alternative oxidase is a respiratory chain protein found in plants, fungi and some parasites that still remains physically uncharacterised. In this report we present EPR evidence from parallel mode experiments which reveal signals at approximately g  = 16 in both purified alternative oxidase protein (g  = 16.9), isolated mitochondrial membranes (g  = 16.1), and in trypanosomal AOX expressed in Escherichia coli membranes (g  = 16.4). Such signals are indicative of a dicarboxylate diiron centre at the active site of the enzyme. To our knowledge these data represent the first EPR signals from AOX present in its native environment.
Keywords: Alternative oxidase; Diiron; Electron transfer; Respiration; Monotopic integral membrane protein;

The influence of the histidine axial ligand to the PD1 chlorophyll of photosystem II on the redox potential and spectroscopic properties of the primary electron donor, P680, was investigated in mutant oxygen-evolving photosystem II (PSII) complexes purified from the thermophilic cyanobacterium Thermosynechococcus elongatus. To achieve this aim, a mutagenesis system was developed in which the psbA 1 and psbA 2 genes encoding D1 were deleted from a His-tagged CP43 strain (to generate strain WT) and mutations D1-H198A and D1-H198Q were introduced into the remaining psbA 3 gene. The O2-evolving activity of His-tagged PSII isolated from WT was found to be significantly higher than that measured from His-tagged PSII isolated from WT in which psbA 1 is expected to be the dominantly expressed form. PSII purified from both the D1-H198A and D1-H198Q mutants exhibited oxygen-evolving activity as high as that from WT. Surprisingly, a variety of kinetic and spectroscopic measurements revealed that the D1-H198A and D1-H198Q mutations had little effect on the redox and spectroscopic properties of P680, in contrast to the earlier results from the analysis of the equivalent mutants constructed in Synechocystis sp. PCC 6803 [B.A. Diner, E. Schlodder, P.J. Nixon, W.J. Coleman, F. Rappaport, J. Lavergne, W.F. Vermaas, D.A. Chisholm, Site-directed mutations at D1-His198 and D2-His197 of photosystem II in Synechocystis PCC 6803: sites of primary charge separation and cation and triplet stabilization, Biochemistry 40 (2001) 9265–9281]. We conclude that the nature of the axial ligand to PD1 is not an important determinant of the redox and spectroscopic properties of P680 in T. elongatus.
Keywords: Photosystem II; P680; Electron transfer; Chlorophyll axial ligand; Site-directed mutagenesis; Thermosynechococcus elongatus;

Deuterium isotope effect of proton pumping in cytochrome c oxidase by Lina Salomonsson; Gisela Brändén; Peter Brzezinski (343-350).
In mitochondria and many aerobic bacteria cytochrome c oxidase is the terminal enzyme of the respiratory chain where it catalyses the reduction of oxygen to water. The free energy released in this process is used to translocate (pump) protons across the membrane such that each electron transfer to the catalytic site is accompanied by proton pumping. To investigate the mechanism of electron–proton coupling in cytochrome c oxidase we have studied the pH-dependence of the kinetic deuterium isotope effect of specific reaction steps associated with proton transfer in wild-type and structural variants of cytochrome c oxidases in which amino-acid residues in proton-transfer pathways have been modified. In addition, we have solved the structure of one of these mutant enzymes, where a key component of the proton-transfer machinery, Glu286, was modified to an Asp. The results indicate that the P3  → F3 transition rate is determined by a direct proton-transfer event to the catalytic site. In contrast, the rate of the F3  → O4 transition, which involves simultaneous electron transfer to the catalytic site and is characteristic of any transition during CytcO turnover, is determined by two events with similar rates and different kinetic isotope effects. These reaction steps involve transfer of protons, that are pumped, via a segment of the protein including Glu286 and Arg481.
Keywords: Cytochrome aa3; Electron transfer; Proton transfer; Membrane protein; Transmembrane transport; Kinetics; Spectroscopy; Mitochondria;

Photosystem I complexes associated with fucoxanthin-chlorophyll-binding proteins from a marine centric diatom, Chaetoceros gracilis by Yohei Ikeda; Masayuki Komura; Mai Watanabe; Chie Minami; Hiroyuki Koike; Shigeru Itoh; Yasuhiro Kashino; Kazuhiko Satoh (351-361).
Diatoms occupy a key position as a primary producer in the global aquatic ecosystem. We developed methods to isolate highly intact thylakoid membranes and the photosystem I (PS I) complex from a marine centric diatom, Chaetoceros gracilis. The PS I reaction center (RC) was purified as a super complex with light-harvesting fucoxanthin-chlorophyll (Chl)-binding proteins (FCP). The super complex contained 224 Chl a, 22 Chl c, and 55 fucoxanthin molecules per RC. The apparent molecular mass of the purified FCP–PS I super complex (∼ 1000 kDa) indicated that the super complex was composed of a monomer of the PS I RC complex and about 25 copies of FCP. The complex contained menaquinone-4 as the secondary electron acceptor A1 instead of phylloquinone. Time-resolved fluorescence emission spectra at 77 K indicated that fast (16 ps) energy transfer from a Chl a band at 685 nm on FCP to Chls on the PS I RC complex occurs. The ratio of fucoxanthin to Chl a on the PS I-bound FCP was lower than that of weakly bound FCP, suggesting that PS I-bound FCP specifically functions as the mediator of energy transfer between weakly bound FCPs and the PS I RC.
Keywords: Chaetoceros gracilis; Diatom; Fucoxanthin-chlorophyll-binding protein; Photosystem I; Time-resolved fluorescence spectra;

Inhibition of the ATPase activity of the catalytic portion of ATP synthases by cationic amphiphiles by Manuel J. Datiles; Eric A. Johnson; Richard E. McCarty (362-368).
Melittin, a cationic, amphiphilic polypeptide, has been reported to inhibit the ATPase activity of the catalytic portions of the mitochondrial (MF1) and chloroplast (CF1) ATP synthases. Gledhill and Walker [J.R. Gledhill, J.E. Walker. Inhibition sites in F1-ATPase from bovine heart mitochondria, Biochem. J. 386 (2005) 591–598.] suggested that melittin bound to the same site on MF1 as IF1, the endogenous inhibitor polypeptide. We have studied the inhibition of the ATPase activity of CF1 and of F1 from Escherichia coli (ECF1) by melittin and the cationic detergent, cetyltrimethylammonium bromide (CTAB). The Ca2+- and Mg2+-ATPase activities of CF1 deficient in its inhibitory ε subunit (CF1-ε) are sensitive to inhibition by melittin and by CTAB. The inhibition of Ca2+-ATPase activity by CTAB is irreversible. The Ca2+-ATPase activity of F1 from E. coli (ECF1) is inhibited by melittin and the detergent, but Mg2+-ATPase activity is much less sensitive to both reagents. The addition of CTAB or melittin to a solution of CF1-ε or ECF1 caused a large increase in the fluorescence of the hydrophobic probe, N-phenyl-1-naphthylamine, indicating that the detergent and melittin cause at least partial dissociation of the enzymes. ATP partially protects CF1-ε from inhibition by CTAB. We also show that ATP can cause the aggregation of melittin. This result complicates the interpretation of experiments in which ATP is shown to protect enzyme activity from inhibition by melittin. It is concluded that melittin and CTAB cause at least partial dissociation of the α/β heterohexamer.
Keywords: Chloroplast ATP synthase; ATPase activity; Melittin; Cationic detergent; Subunit dissociation;

Oxygen evolution in the thylakoid-lacking cyanobacterium Gloeobacter violaceus PCC 7421 by Kohei Koyama; Hiroyuki Suzuki; Takumi Noguchi; Seiji Akimoto; Tohru Tsuchiya; Mamoru Mimuro (369-378).
The oxygen-evolving reactions of the thylakoid-lacking cyanobacterium Gloeobacter violaceus PCC 7421 were compared with those of Synechocystis sp. PCC 6803. Four aspects were considered: sequence conservation in three extrinsic proteins for oxygen evolution, steady-state oxygen-evolving activity, charge recombination reactions, i.e., thermoluminescence and oscillation patterns of delayed luminescence on a second time scale and delayed fluorescence on the nanosecond time scale at − 196 °C. Even though there were significant differences between the amino acid sequences of extrinsic proteins in G. violaceus and Synechocystis sp. PCC 6803, the oxygen-evolving activities were similar. The delayed luminescence oscillation patterns and glow curves of thermoluminescence were essentially identical between the two species, and the nanosecond delayed fluorescence spectral profiles and lifetimes were also very similar. These results indicate clearly that even though the oxygen-evolving reactions are carried out in the periplasm by components with altered amino acid sequences, the essential reaction processes for water oxidation are highly conserved. In contrast, we observed significant changes on the reduction side of photosystem II. Based on these data, we discuss the oxygen-evolving activity of G. violaceus.
Keywords: Cyanobacteria; Extrinsic protein; Oxygen evolution; Photosystem II; Delayed luminescence; Gloeobacter violaceus PCC 7421;

Photosystems must efficiently dissipate absorbed light energy under freezing conditions. To clarify the energy dissipation mechanisms, we examined energy transfer and dissipation dynamics in needles of the evergreen plant Taxus cuspidata by time-resolved fluorescence spectroscopy. In summer and autumn, the energy transfer processes were similar to those reported in other higher plants. However, in winter needles, fluorescence lifetimes became shorter not only in PSII but also in PSI, indicating energy dissipation in winter needles. In addition, almost the same fluorescence spectra were obtained with different excitation wavelengths. In contrast, the fluorescence spectrum showed a large difference due to excitation wavelength in spring needles. The fluorescence spectrum of spring needles in 550-nm excitation showed similar spectra to that of winter needles, however, red-chlorophyll fluorescence was not observed in chlorophyll excitation. These observations suggest that some complexes with some kind of red-shifted carotenoid and red-chlorophyll unlink from the core complex in spring. Seasonal changes of excitation energy dynamics are also discussed in relation to changes in thylakoid stacking.
Keywords: Seasonal change; Energy transfer; Quench; Time-resolved fluorescence; Evergreen plant; Thylakoid stacking;

In Chlamydomonas, the loss of ND5 subunit prevents the assembly of whole mitochondrial complex I and leads to the formation of a low abundant 700 kDa subcomplex by Pierre Cardol; Layla Boutaffala; Samy Memmi; Bart Devreese; René Fernand Matagne; Claire Remacle (388-396).
In the green alga Chlamydomonas reinhardtii, a mutant deprived of complex I enzyme activity presents a 1T deletion in the mitochondrial nd5 gene. The loss of the ND5 subunit prevents the assembly of the 950 kDa whole complex I. Instead, a low abundant 700 kDa subcomplex, loosely associated to the inner mitochondrial membrane, is assembled. The resolution of the subcomplex by SDS-PAGE gave rise to 19 individual spots, sixteen having been identified by mass spectrometry analysis. Eleven, mainly associated to the hydrophilic part of the complex, are homologs to subunits of the bovine enzyme whereas five (including gamma-type carbonic anhydrase subunits) are specific to green plants or to plants and fungi. None of the subunits typical of the β membrane domain of complex I enzyme has been identified in the mutant. This allows us to propose that the truncated enzyme misses the membrane distal domain of complex I but retains the proximal domain associated to the matrix arm of the enzyme. A complex I topology model is presented in the light of our results. Finally, a supercomplex most probably corresponding to complex I–complex III association, was identified in mutant mitochondria, indicating that the missing part of the enzyme is not required for the formation of the supercomplex.
Keywords: Chlamydomonas mitochondria; ND5 subunit; Complex I mutant; NADH:Ubiquinone oxidoreductase;