BBA - Bioenergetics (v.1506, #2)

Analysis of suppressor mutation reveals long distance interactions in the bc 1 complex of Saccharomyces cerevisiae by Gaël Brasseur; Jean-Paul Di Rago; Piotr P. Slonimski; Danielle Lemesle-Meunier (89-102).
Four totally conserved glycines are involved in the packing of the two cytochrome b hemes, b L and b H, of the bc 1 complex. The conserved glycine 131 is involved in the packing of heme b L and is separated by only 3 Å from this heme in the bc 1 complex structure. The cytochrome b respiratory deficient mutant G131S is affected in the assembly of the bc 1 complex. An intragenic suppressor mutation was obtained at position 260, in the ef loop, where a glycine was replaced by an alanine. This respiratory competent revertant exhibited a low bc 1 complex activity and was affected in the electron transfer at the QP site. The k min for the substrate DBH2 was diminished by an order of magnitude and EPR spectra showed a partially empty QP site. However, the binding of the QP site inhibitors stigmatellin and myxothiazol remained unchanged in the suppressor strain. Optical spectroscopy revealed that heme b L is red shifted by 0.8 nm and that the E m of heme b L was slightly increased (+20 mV) in the revertant strain as compared to wild type strain values. Addition of a methyl group at position 260 is thus sufficient to allow the assembly of the bc 1 complex and the insertion of heme b L despite the presence of the serine at position 131. Surprisingly, reversion at position 260 was located 13 Å away from the original mutation and revealed a long distance interaction in the yeast bc 1 complex.
Keywords: bc 1 complex; Revertant; Long distance interaction; Heme b L; Quinol binding; Saccharomyces cerevisiae;

The Cbp3 protein of Saccharomyces cerevisiae is an enzyme-specific chaperone required for the assembly of ubiquinol-cytochrome c reductase of the mitochondrial respiratory chain. To gain preliminary insight into the role of Cbp3p during assembly, 29 independently isolated mutants were examined to define functional regions of the protein. Mutants were analyzed with respect to respiratory growth, ubiquinol-cytochrome c reductase assembly, and steady state amounts of enzyme subunits and Cbp3p. Three regions essential for Cbp3p activity were identified: regions 1 and 3 were required for Cbp3p function, while region 2 was necessary for protein stability. Mutation of Glu134 in region 1 (Cys124 through Ala140) impaired the ability of the Rieske FeS protein to assemble with the enzyme complex. Mutations targeted to region 3 (Gly223 through Asp229) primarily affected the 14 kDa subunit and cytochrome c 1 assembly. Gly223 was found especially sensitive to mutation and the introduction of charged residues at this site compromised Cbp3p functional activity. Region 2 (Leu167 through Pro175) overlapped the single hydrophobic domain of Cbp3p. Mutations within this area altered the association of Cbp3p with the mitochondrial membrane resulting in enhanced protein turnover. The role of the amino-terminus in Cbp3p activity was investigated using cbp3 deletion strains Δ12–23, Δ24–54, Δ56–96 and Δ12–96. All mutants were respiratory competent, indicating that residues 12–96 were not essential for Cbp3p function, stability or mitochondrial import. Analysis of carboxy-terminal deletion mutants demonstrated that the final 44 residues were not necessary for Cbp3p function; however, alterations in the secondary structure of the extreme carboxy-terminal 17 residues affected assembly protein activity.
Keywords: Organelle biogenesis; Respiratory chain; Chaperone-assisted assembly of multisubunit enzyme;

The catalytic Mn cluster of the photosynthetic oxygen-evolving system is oxidized via a tyrosine, YZ, by a photooxidized chlorophyll a moiety, P+ 680. The rapid reduction of P+ 680 by YZ in nanoseconds requires the intactness of an acid/base cluster around YZ with an apparent functional pK of <5. The removal of Mn (together with bound Ca) shifts the pK of the acid/base cluster from the acid into the neutral pH range. At alkaline pH the electron transfer (ET) from YZ to P+ 680 is still rapid (<1 μs), whereas at acid pH the ET is much slower (10–100 μs) and steered by proton release. In the intermediate pH domain one observes a mix of these kinetic components (see R. Ahlbrink, M. Haumann, D. Cherepanov, O. Bögershausen, A. Mulkidjanian, W. Junge, Biochemistry 37 (1998)). The overall kinetics of P680 + reduction by YZ in Mn-depleted photosystem II (PS II) has been previously shown to be slowed down by divalent cations (added at >10 μM), namely: Mn2+, Co2+, Ni2+, Cu2+, Zn2+ (C.W. Hoganson, P.A. Casey, O. Hansson, Biochim. Biophys. Acta 1057 (1991)). Using Mn-depleted PS II core particles from pea as starting material, we re-investigated this phenomenon at nanosecond resolution, aiming at the effect of divalent cations on the particular kinetic components of P+ 680 reduction. To our surprise we found only the slower, proton steered component retarded by some added cations (namely Co2+/Zn2+>Fe2+>Mn2+). Neither the fast component nor the apparent pK of the acid/base cluster around YZ was affected. Apparently, the divalent cations acted (electrostatically) on the proton release channel that connects the oxygen-evolving complex with the bulk water, but not on the ET between YZ and P+ 680, proper. Contrastingly, Ca2+ and Mg2+, when added at >5 mM, accelerated the slow component of P+ 680 reduction by YZ and shifted the apparent pK of YZ from 7.4 to 6.6 and 6.7, respectively. It was evident that the binding site(s) for added Ca2+ and Mg2+ were close to YZ proper. The data obtained are discussed in relation to the nature of the metal-binding sites in photosystem II.
Keywords: Photosystem II; Water oxidation; Tyrosine Z; Proton coupled electron transfer; H/2H isotope effect; Hydrogen bond;

Quinolones and their N-oxides as inhibitors of photosystem II and the cytochrome b 6/f-complex by Ellen Reil; Gerhard Höfle; Wilfried Draber; Walter Oettmeier (127-132).
4(1H)-quinolones (2-alkyl- (1), 2-alkyl-3-methyl- (2), 2-methyl-3-alkyl- (3), 1-hydroxy-2-methyl-3-alkyl- (4) and 1-hydroxy-2-alkyl- (5)) with n-alkyl side chains varying from C5 to C17 have been synthesized and tested for biological activity in photosystem II and the cytochrome b 6/f-complex. In photosystem II, quinolones 1 and 2 showed only moderate activity, whereas 3<5<4 (increasing activity) were potent inhibitors. Displacement experiments with [14C]atrazine indicated that the quinolones share an identical binding site with other photosystem II commercial herbicides. In the cytochrome b 6/f-complex, only 3<4 showed enhanced activity. Maximal inhibitory potency was achieved at a carbon chain length of 12–14 Å. Further increase of the chain length decreased activity. In a quantitative structure–activity relationship inhibitory activity in photosystem II and the cytochrome b 6/f-complex could be correlated to the physicochemical parameters lipophilicity π and/or to STERIMOL L.
Keywords: Structure–activity relationship; QB-site; [14C]Atrazine displacement; Heterocyclic ketone;

The plastoquinone pool during dark adaptation is reduced by endogenous reductants and oxidized at the expense of molecular oxygen. We report here on the redox state of plastoquinone in darkness, using as an indicator the chlorophyll fluorescence kinetics of whole cells of a Chlamydomonas reinhardtii mutant strain lacking the cytochrome b 6 f complex. When algae were equilibrated with a mixture of air and argon at 1.45% air, plastoquinol oxidation was inhibited whereas mitochondrial respiration was not. Consequently, mitochondrial oxidases cannot be responsible for the oxygen consumption linked to plastoquinol oxidation. Plastoquinol oxidation in darkness turned out to be sensitive to n-propyl gallate (PG) and insensitive to salicylhydroxamic acid (SHAM), whereas mitochondrial respiration was sensitive to SHAM and PG. Thus, both PG treatment and partial anaerobiosis allow to draw a distinction between an inhibition of plastoquinol oxidation and an inhibition of mitochondrial respiration, indicating the presence of a plastoquinol:oxygen oxidoreductase. The possible identification of this oxidase with an oxidase involved in carotenoid biosynthesis is discussed in view of various experimental data.
Keywords: Chlorophyll fluorescence kinetics; Chlororespiration; Plastoquinol:oxygen oxidoreductase; n-Propyl gallate; Carotenoid biosynthesis; Chlamydomonas reinhardtii;