BBA - Bioenergetics (v.1608, #1)

Guide for Authors (vii-xii).

The gross structure of the respiratory complex I: a Lego System by Thorsten Friedrich; Bettina Böttcher (1-9).
The proton-pumping NADH:ubiquinone oxidoreductase, also called complex I, is the entry point for electrons into the respiratory chains of many bacteria and mitochondria of most eucaryotes. It couples electron transfer with the translocation of protons across the membrane, thus providing the proton motive force essential for energy-consuming processes. Electron microscopy revealed the ‘L’-shaped structure of the bacterial and mitochondrial complex with two arms arranged perpendicular to each other. Recently, we showed that the Escherichia coli complex I takes on another stable conformation with the two arms arranged side by side resulting in a horseshoe-shaped structure. This model reflects the evolution of complex I from pre-existing modules for electron transfer and proton translocation.
Keywords: NADH:ubiquinone oxidoreductase; Complex I; Electron microscopy; Single particle analysis; Image processing; Protein structure; Modular evolution;

Relatively little is known about the functions of specific molecular interactions between membrane proteins and membrane lipids. The structural and functional consequences of disrupting a previously identified interaction between a molecule of the diacidic lipid cardiolipin and the purple bacterial reaction centre were examined. Mutagenesis of a highly conserved arginine (M267) that is responsible for binding the head-group of the cardiolipin (to leucine) did not affect the rate of photosynthetic growth, the functional properties of the reaction centre, or the X-ray crystal structure of the complex (determined to a resolution of 2.8 Å). However, the thermal stability of the protein was compromised by this mutation, part of the reaction centre population showing an approximately 5 °C decrease in melting temperature in response to the arginine to leucine mutation. The crystallised mutant reaction centre also no longer bound detectable amounts of cardiolipin at this site. Taken together, these observations suggest that this particular protein–lipid interaction contributes to the thermal stability of the complex, at least when in detergent micelles. These findings are discussed in the light of proposals concerning the unfolding processes that occur when membrane proteins are heated, and we propose that one function of the cardiolipin is to stabilise the interaction between adjacent membrane-spanning α-helices in a region where there are no direct protein–protein interactions.
Keywords: Membrane protein; Membrane lipid; Purple bacterial reaction centre;

Biocompatible nanosized polyamidoamine (PAMAM) dendrimer films provided a suitable microenvironment for heme proteins to transfer electron directly with underlying pyrolytic graphite (PG) electrodes. Hemoglobin (Hb), myoglobin (Mb), horseradish peroxidase (HRP), and catalase (Cat) incorporated in PAMAM films exhibited a pair of well-defined, quasi-reversible cyclic voltammetric peaks, respectively, characteristic of the protein heme Fe(III)/Fe(II) redox couples. While Hb-, Mb-, and HRP-PAMAM films showed the cyclic voltammetry (CV) peaks at about −0.34 V vs. saturated calomel electrode (SCE) in pH 7.0 buffers, Cat-PAMAM films displayed the peak pair at a more negative potential of −0.47 V. The protein-PAMAM films demonstrated a surface-confined or thin-layer voltammetric behavior. The electrochemical parameters such as apparent heterogeneous electron transfer rate constants (k s) and formal potentials (E°′) were estimated by square wave voltammetry with nonlinear regression analysis. UV–vis and IR spectroscopy showed that the proteins retained their near-native secondary structures in PAMAM films. Oxygen, hydrogen peroxide, and nitrite were catalytically reduced at the protein-PAMAM film electrodes, showing the potential applicability of the films as the new type of biosensors or bioreactors based on direct electrochemistry of the proteins.
Keywords: Myoglobin; Hemoglobin; Horseradish peroxidase; Catalase; Polyamidoamine dendrimer; Direct electrochemistry; Electrochemical catalysis;

Energy transfer in monomeric phycoerythrocyanin by Peter Zehetmayer; Michaela Kupka; Hugo Scheer; Andreas Zumbusch (35-44).
Phycoerythrocyanin (PEC) is part of the phycobilisome of cyanobacteria. Its monomer carries one phycoviolobilin and two phycocyanobilins (PCB) as chromophores. For an understanding of the complicated energy transfer in phycobilisomes, a detailed knowledge of the processes in the constituting building proteins is indispensable. We report the experimental data necessary for the description of Förster energy transfer in monomeric PEC, including fluorescence lifetimes and quantum yields of the two subunits. The bulk experiments are complemented by studies of single PEC molecules. Förster energy calculations and Monte Carlo simulations based on the bulk data are presented. They reveal that earlier experimental findings of energy transfer heterogeneities in single PEC molecules originate in spectral shifts between the contributing chromophores.
Keywords: Phycoerythrocyanin; Biliprotein; Light harvesting; Energy transfer; Single molecule; Photosynthesis;

The main external alternative NAD(P)H dehydrogenase of Neurospora crassa mitochondria by Patrı́cia Carneiro; Margarida Duarte; Arnaldo Videira (45-52).
A DNA sequence homologous to non-proton-pumping NADH dehydrogenase genes was found in the genome of Neurospora crassa encoding a polypeptide of 577 amino acid residues, molecular mass of 64,656 Da, with a putative transmembrane domain. Analysis of fungal mitochondria fractionated with digitonin indicates that the protein is located at the outer face of the inner membrane of the organelle (external enzyme). The corresponding gene was inactivated by the generation of repeat-induced point mutations. Mitochondria from the resulting null-mutant nde2 are highly deficient in the oxidation of cytosolic NADH and NADPH. A triple mutant nde1/nde2/ndi1, lacking mitochondrial alternative NAD(P)H dehydrogenases, was obtained, indicating that these proteins are not essential in N. crassa. However, crosses between the nde2 mutant strain and complex I-deficient mutants yielded no viable double mutants. Transcription of the nde-2 gene, as well as of ndi-1 (internal enzyme), is repressed in the late exponential phase of fungal growth.
Keywords: Mitochondrion; NADH dehydrogenase; Respiratory mutant; Gene expression; Neurospora crassa;

Quantification of photosystem I and II in different parts of the thylakoid membrane from spinach by Ravi Danielsson; Per-Åke Albertsson; Fikret Mamedov; Stenbjörn Styring (53-61).
Electron paramagnetic resonance (EPR) was used to quantify Photosystem I (PSI) and PSII in vesicles originating from a series of well-defined but different domains of the thylakoid membrane in spinach prepared by non-detergent techniques. Thylakoids from spinach were fragmented by sonication and separated by aqueous polymer two-phase partitioning into vesicles originating from grana and stroma lamellae. The grana vesicles were further sonicated and separated into two vesicle preparations originating from the grana margins and the appressed domains of grana (the grana core), respectively. PSI and PSII were determined in the same samples from the maximal size of the EPR signal from P700+ and YD , respectively. The following PSI/PSII ratios were found: thylakoids, 1.13; grana vesicles, 0.43; grana core, 0.25; grana margins, 1.28; stroma lamellae 3.10. In a sub-fraction of the stroma lamellae, denoted Y-100, PSI was highly enriched and the PSI/PSII ratio was 13. The antenna size of the respective photosystems was calculated from the experimental data and the assumption that a PSII center in the stroma lamellae (PSIIβ) has an antenna size of 100 Chl. This gave the following results: PSI in grana margins (PSIα) 300, PSI (PSIβ) in stroma lamellae 214, PSII in grana core (PSIIα) 280. The results suggest that PSI in grana margins have two additional light-harvesting complex II (LHCII) trimers per reaction center compared to PSI in stroma lamellae, and that PSII in grana has four LHCII trimers per monomer compared to PSII in stroma lamellae. Calculation of the total chlorophyll associated with PSI and PSII, respectively, suggests that more chlorophyll (about 10%) is associated with PSI than with PSII.
Keywords: Photosystem I; Photosystem II; Thylakoid membrane; Photosystem antennae; EPR; Phase partition;

Electron spin resonance spectroscopy and liquid chromatography have been used to detect radical formation and fragmentation of polypeptides during photoinhibition of purified major antenna proteins, free of protease contaminants. In the absence of oxygen and light, no radicals were observed and there was no damage to the proteins. Similarly illumination of the apoproteins did not induce any polypeptide fragmentation, suggesting that chlorophyll, light and atmospheric oxygen are all participating in antenna degradation. The use of TEMP and DMPO as spin traps showed that protein damage initiates with generation of 1O2, presumably from a triplet chlorophyll, acting as a Type II photosensitizer which attacks directly the amino acids causing a complete degradation of protein into small fragments, without the contribution of proteases. Through the use of scavengers, it was shown that superoxide and H2O2 were not involved initially in the reaction mechanism. A higher production of radicals was observed in trimers than in monomeric antenna, while radical production is strongly reduced when antennae were organized in the photosystem II (PSII) complex. Thus, monomerization of antennae as well as their incorporation into the PSII complex seem to represent physiologically protected forms. A comparison is made of the photoinhibition mechanisms of different photosynthetic systems.
Keywords: Reactive oxygen species; Light-harvesting complex; ESR; Protein degradation; Singlet oxygen; Photoinhibition;