BBA - Bioenergetics (v.1657, #2-3)

Allophycocyanin and energy transfer by Robert MacColl (73-81).
Allophycocyanin is a biliprotein located in the core of the phycobilisome. The biliprotein is isolated and purified as a trimer (α3β3), where a monomer is an αβ structure. Each α and β subunit has a single noncyclic tetrapyrrole chromophore, called phycocyanobilin. The trimer of allophycocyanin has an unusual absorption maximum at 650 nm with a shoulder at 620 nm, while the monomer has an absorption maximum at 615 nm. Two explanations have been proposed for the 650-nm maximum. In one, this maximum is produced by the interaction of a particular local protein environment for three of the chromophores, causing them to red shift, while the other three chromophores are at a higher energy. Energy is transferred from the high- to the low-energy chromophores by Förster resonance energy transfer, the donor–acceptor model. In the second proposal, there is strong exciton coupling between two chromophores of the trimer that closely approach across the monomer–monomer interface. The strong interaction causes exciton splitting and a red shift in the absorption. There are three of these strongly coupled chromophore pairs, and energy is transferred between the two-exciton states of a pair by internal conversion. A variety of biophysical methods have been used to examine this question. Although evidence supporting both models has been produced, sophisticated ultra fast fluorescence results from a plethora of approaches now firmly point to the latter strong coupling hypothesis as being more likely. Between the different strongly coupled pairs, Förster resonance energy transfer should occur. For monomers of allophycocyanin, Förster resonance energy transfer occurs between the two chromophores.
Keywords: Allophycocyanin; Biliprotein; Exciton coupling; Femtosecond fluorescence spectroscopy; Förster resonance energy transfer; Phycobilisome; Internal conversion; Photosynthesis;

Global and target analysis of time-resolved spectra by Ivo H.M. van Stokkum; Delmar S. Larsen; Rienk van Grondelle (82-104).
In biological/bioenergetics research the response of a complex system to an externally applied perturbation is often studied. Spectroscopic measurements at multiple wavelengths are used to monitor the kinetics. These time-resolved spectra are considered as an example of multiway data. In this paper, the methodology for global and target analysis of time-resolved spectra is reviewed. To fully extract the information from the overwhelming amount of data, a model-based analysis is mandatory. This analysis is based upon assumptions regarding the measurement process and upon a physicochemical model for the complex system. This model is composed of building blocks representing scientific knowledge and assumptions. Building blocks are the instrument response function (IRF), the components of the system connected in a kinetic scheme, and anisotropy properties of the components. The combination of a model for the kinetics and for the spectra of the components results in a more powerful spectrotemporal model. The model parameters, like rate constants and spectra, can be estimated from the data, thus providing a concise description of the complex system dynamics. This spectrotemporal modeling approach is illustrated with an elaborate case study of the ultrafast dynamics of the photoactive yellow protein.
Keywords: Global analysis; Multiway data; Photoactive yellow protein; Spectrotemporal model; Target analysis; Time-resolved spectroscopy;

Subcellular localization of VDAC in mitochondria and ER in the cerebellum by Varda Shoshan-Barmatz; Ran Zalk; Dan Gincel; Noga Vardi (105-114).
The voltage-dependent anion channel (VDAC) provides passage for adenine nucleotides, Ca2+ and other metabolites into and from mitochondria. Here, the intracellular localization and oligomeric organization of VDAC in brain mitochondria and ER are demonstrated. Immunohistochemical staining of VDAC in rat cerebellum showed high labeling of the Purkinje neurons. Immunogold labeling and EM analysis of the cerebellar molecular layer showed specific VDAC immunostaining of the mitochondrial outer membrane, highly enhanced in contact sites between mitochondria or between mitochondria and associated ER. Purified ER membranes contain VDAC, but not other mitochondrial proteins. Chemical cross-linking of isolated mitochondria, ER or purified VDAC demonstrated the existence of VDAC in oligomeric form. Based on the enrichment of VDAC in the junctional face of closely associated mitochondrial and ER membranes and the existence of VDAC oligomers, we propose an involvement of VDAC in specialized intermembrane communication between mitochondria or between ER and mitochondria, serving to complement the tight structural and functional coupling observed between these organelles.
Keywords: VDAC; ER and mitochondria; Cerebellum;

Redox properties of Arabidopsis cytochrome c 6 are independent of the loop extension specific to higher plants by Jürgen Wastl; Fernando P Molina-Heredia; Manuel Hervás; José A Navarro; Miguel A De la Rosa; Derek S Bendall; Christopher J Howe (115-120).
Cytochrome c 6 (cytc 6) from Arabidopsis differs from the cyanobacterial and algal homologues in several redox properties. It is possible that these differences might be due to the presence of a 12 amino acid residue loop extension common to higher plant cytc 6 proteins. However, homology modelling suggests this is not the case. We report experiments to test if differences in biochemical properties could be due to this extension. Analysis of mutant forms of Arabidopsis cytc 6 in which the entire extension was lacking, or a pair of cysteine residues in the extension had been exchanged for serine, revealed no significant effect of these changes on either the redox potential of the haem group or the reactivity towards Photosystem I (PSI). We conclude that the differences in properties are due to more subtle unidentified differences in structure, and that the sequence extension in the higher plant proteins has a function yet to be identified.
Keywords: Cytochrome c 6; Arabidopsis; Protein expression; Spectroscopy; Redox midpoint potential; Laser flash spectroscopy;

The action of various inhibitors affecting the donor and acceptor sides of photosystem II (PSII) on the polyphasic rise of chlorophyll (Chl) fluorescence was studied in thylakoids isolated from pea leaves. Low concentrations of diuron and stigmatellin increased the magnitude of J-level of the Chl fluorescence rise. These concentrations barely affected electron transfer from PSII to PSI as revealed by the unchanged magnitude of the fast component (t 1/2=24 ms) of P700+ dark reduction. Higher concentrations of diuron and stigmatellin suppressed electron transport from PSII to PSI, which corresponded to the loss of thermal phase, the Chl fluorescence rise from J-level to the maximal, P-level. The effect of various concentrations of carbonylcyanide m-chlorophenylhydrazone (CCCP), which abolishes S-state cycle and binds at the plastoquinone site on QB, the secondary quinone acceptor PSII, on the Chl fluorescence rise was very similar to that of diuron and stigmatellin. Low concentrations of diuron, stigmatellin, or CCCP given on the background of N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD), which is shown to initiate the appearance of a distinct I-peak in the kinetics of Chl fluorescence rise measured in isolated thylakoids [BBA 1607 (2003) 91], increased J-step yield to I-step level and retarded Chl fluorescence rise from I-step to P-step. The increased J-step fluorescence rise caused by these three types of inhibitors is attributed to the suppression of the non-photochemical quenching of Chl fluorescence by [S2+ S3] states of the oxygen-evolving complex and oxidized P680, the primary donor of PSII reaction centers. In the contrary, the decreased fluorescence yield at P step (J–P, passing through I) is related to the persistence of a “plastoquinone”-type quenching owing to the limited availability of photochemically generated electron equivalents to reduce PQ pool in PSII centers where the S-state cycle of the donor side is modified by the inhibitor treatments.
Keywords: Chlorophyll fluorescence; Diuron; Photosystem II; Stigmatellin; Thylakoid;

Nonenzymatic chromophore attachment in biliproteins: conformational control by the detergent Triton X-100 by Kai-Hong Zhao; Jing-Ping Zhu; Bo Song; Ming Zhou; Max Storf; Stephan Böhm; Claudia Bubenzer; Hugo Scheer (131-145).
While chromophore attachment to α-subunits of cyanobacterial biliproteins has been studied in some detail, little is known about this process in β-subunits. The ones of phycoerythrocyanin and C-phycocyanin each carry two phycocyanobilin (PCB) chromophores covalently attached to cysteins β84 and β155. The differential nonenzymatic reconstitution of PCB to the apoproteins, PecA, PecB, CpcA and CpcB, as well as to mutant proteins of the β-subunits lacking either one of the two binding cysteins, was studied using overexpression of the respective genes. PCB adds selectively to Cys-84 of CpcA, CpcB, PecA, and PecB, but the bound chromophore has a nonnative configuration, and in the case of CpcA, is partly oxidized to mesobiliverdin (MBV). The oxidation is independent of thiols but can be suppressed by ascorbate. The addition to Cys-β84 is suppressed in the presence of detergents like Triton X-100, in favor of an addition to Cys-β155 yielding the correctly bound chromophore. Triton X-100 also inhibits oxidation of the chromophore during addition to CpcA. The effect of Triton X-100 was studied on the isolated components of the reconstitution system. Absorption, fluorescence and circular dichroism spectra indicate a major conformational change of the chromophore upon addition of the detergent, which probably controls the site selectivity of the addition reaction, and inhibits the oxidation of PCB to MBV.
Keywords: Photosynthesis; Cyanobacteria; Phycobiliprotein; Chromophore; Attachment; Chromophore conformation;

Abnormal cardiac energetics in patients carrying the A3243G mtDNA mutation measured in vivo using phosphorus MR spectroscopy by Raffaele Lodi; Bheeshma Rajagopalan; Andrew M Blamire; Jenifer G Crilley; Peter Styles; Patrick F Chinnery (146-150).
Cardiomyopathy is a frequent cause of morbidity and mortality in patients carrying the A3243G transition in the mitochondrial DNA (mtDNA) tRNALeu(UUR) gene, the most common heteroplasmic single mtDNA defect. We used phosphorus magnetic resonance spectroscopy (31P-MRS) to look for evidence of an in vivo bioenergetics defect in patients carrying the A3243G mtDNA mutation with and without echocardiographic signs of left ventricle hypertrophy (LVH). Eight patients, three with LVH, carrying the A3243G mtDNA mutation and 10 healthy subjects underwent one-dimensional chemical shift imaging 31P-MRS. In the patients, mean cardiac phosphocreatine to adenosine triphosphate ratio (PCr/ATP) (1.55±0.58) was significantly reduced compared to the control group (2.34±0.14; P<0.001). Cardiac PCr/ATP was within the normal range only in one case that showed normal echocardiography. Our results point to a central role of bioenergetics deficit in the development of cardiac hypertrophy in patients with the A3243G mtDNA mutation. Impaired cardiac energy metabolism in patients with normal echocardiography suggests that the enhancement of mitochondrial function may be beneficial not only to patients with cardiac hypertrophy but also to those patients carrying the mutation in the absence of signs of cardiac hypertrophy and/or dysfunction but with cardiac bioenergetics deficit.
Keywords: Magnetic resonance spectroscopy; Metabolism; Cardiomyopathy;

Effects of N-acylethanolamines on mitochondrial energetics and permeability transition by Michał Wasilewski; Mariusz R Więckowski; Dorota Dymkowska; Lech Wojtczak (151-163).
Effects of N-acylethanolamines (NAEs): N-arachidonoylethanolamine (anandamide), N-oleoylethanolamine and N-palmitoylethanolamine, on energy coupling and permeability of rat heart mitochondria were investigated. In nominally Ca2+-free media, these compounds exerted a weak protonophoric effect manifested by dissipation of the transmembrane potential and stimulation of resting state respiration. The strongest action was exhibited by N-arachidonoylethanolamine, followed by N-oleoylethanolamine, whereas N-palmitoylethanolamine was almost inactive. These protonophoric effects were resistant to cyclosporin A (CsA) and were much weaker than those of corresponding nonesterified fatty acids. In uncoupled mitochondria N-arachidonoylethanolamine and N-oleoylethanolamine partly inhibited mitochondrial respiration with glutamate and succinate but not with tetramethyl-p-phenylenediamine (TMPD) plus ascorbate as respiratory substrates. In mitochondria preloaded with small amounts of Ca2+, NAEs produced a much stronger dissipation of the membrane potential and a release of accumulated calcium, both effects being inhibited by CsA, indicative for opening of the mitochondrial permeability transition pore (PTP). Again, the potency of this action was N-arachidonoylethanolamine>N-oleoylethanolamine>N-palmitoylethanolamine. However, in spite of making the matrix space accessible to external [14C]sucrose, N-arachidonoylethanolamine and N-oleoylethanolamine resulted in only a limited swelling of mitochondria and diminished the rate of swelling produced by high Ca2+ load.
Keywords: N-Acylethanolamine; Anandamide; Endocannabinoid; Proton permeability; Permeability transition pore; Calcium uptake; Calcium release; Membrane potential; Mitochondria;

Photosystem I is not solely responsible for oxygen reduction in isolated thylakoids by Sergey Khorobrykh; Maria Mubarakshina; Boris Ivanov (164-167).
It was found that the contribution of segments of photosynthetic electron transport chain (PETC) besides Photosystem I (PSI) to oxygen reduction increased with increase in light intensity, and at high intensities achieved 50% at pH 5.0, and was higher than 60% at pH 6.5 and pH 7.8. The data are explained as the result of O2 reduction in plastoquinone (PQ) pool as well as in PSI followed by reduction of superoxide radicals generated in both processes by plastohydroquinone.
Keywords: Photoreduction of oxygen; Thylakoid; Plastoquinone pool;