Electron transfer reactions of high-potential cytochromes in the reaction centre of Chromatium tepidum

In the thermophilic purple bacterium C. tepidum, the reaction centre (RC) has a bound cytochrome, containing two high-potential hemes (E_m above +350 mV) and two low-potential hemes (E_m below +150 mV), which re-reduces the photooxidized primary donor, P⁺. We have studied the effects of ambient redox potential and of temperature on the kinetics of that reaction by kinetic flash absorption spectroscopy in chromatophores and isolated reaction centers. When both high-potential hemes are reduced prior to excitation by a short flash of light, the halftime increases slightly between 294 K (t_1/2 = 500 ns) and 217 K (t_1/2 = 1040 ns) indicating an activation energy of 5.0 kJ mol^–1. The fraction of P⁺ which decays by this fast reaction decreases rather steeply around 220 K from nearly 100% at 294 K to nearly 0% below 190 K where P⁺ decays slowly (t_1/2 2.5 ms), probably by return of an electron from the quinone acceptors. When the high-potential hemes are partially oxidized prior to the flash, an additional kinetic phase having a halftime of 30 µs at 294 K is observed. The fractions of RCs that give rise to the individual kinetic phases of P⁺ reduction have been monitored as a function of redox potential. The results can be interpreted in terms of two high-potential hemes which have similar midpoint potentials of +380 ±10 mV and a weak electrostatic interaction.     

Light-induced conformational change at low temperature in the coordination of heme c-556 in the reaction center of Rhodopseudomonas viridis

Isolated reaction centers of Rhodopseudomonas viridis with the two high-potential hemes reduced were illuminated at 5 K. Difference spectra show a bleaching of the heme c-556 alpha bands and a red shift of the Soret band. These effects are reversed by warming to around 80 K. They are not induced by near infra-red light absorbed by the chlorine pigments of the reaction centers and they are not associated with electron transfer from P to Q_A. It is concluded that, following direct excitation, heme c-556 becomes five-coordinated. We find no evidence of a significant photooxidation of heme c-559 under the same conditions.     

Electron transfer between cytochrome c2 and the tetraheme cytochrome c in Rhodopseudomonas viridis

Kinetics of electron transfer from soluble cytochrome c₂ to the tetraheme cytochrome c have been measured in isolated reaction centers and in membrane fragments of the photosynthetic purple bacterium Rhodopseudomonas viridis by time-resolved flash absorption spectroscopy. Absorbance changes kinetics in the region of cytochrome -bands (540–560 nm) were measured at 21 °C under redox conditions where the two high-potential hemes (c-559 and c-556) of the tetraheme cytochrome were chemically reduced. After flash excitation, the heme c-559 donates an electron to the special pair of bacteriochlorophylls and is then re-reduced by heme c-556. The data show that oxidized heme c-556 is subsequently re-reduced by electron transfer from reduced cytochrome c₂ present in the solution. The rate of this reaction has a non-linear dependence on the concentration of cytochrome c₂, suggesting a (minimal) two-step mechanism involving the f ormation of a complex between cytochrome c₂ and the reaction center, followed by intracomplex electron transfer. To explain the monophasic character of the reaction kinetics, we propose a collisional mechanism where the lifetime of the temporary complex is short compared to electron transfer. The limit of the halftime of the bimolecular process when extrapolated to high concentrations of cytochrome c₂ is 60 ± 20 s. There is a large ionic strength effect on the kinetics of electron transfer from cytochrome c₂ to heme c-556. The pseudofirst-order rate constant decreases from 1.1 × 10⁷ M^-1 s^-1 to 1.3 × 10⁶ M^-1 s^-1 when the ionic strength is increased from 1 to 1000 mM. The maximum rate (1.1 × 10⁷ M^-1 s^-1) was obtained at about 1 mM ionic strength. This dependence of the rate on ionic strength s uggests that attractive electrostatic interactions contribute to the binding of cytochrome c₂ with the tetraheme cytochrome. On the basis of our data and of previous molecular modelling, it is proposed that cytochrome c₂ docks close to the low-potential heme c-554 and reduces heme c-556 via c-554.     

Contribution of male-produced proteins to vitellogenesis in Melanoplus sanguinipes

The transfer during copulation of radioactively labelled male accessory reproductive gland (ARG) protein and its accumulation by the ovary of Melanoplus sanguinipes have been studied. Most of the transferred material leaves the spermatheca within 24 hr and enters the haemolymph from which it can be accumulated by the ovary. Injection of labelled male ARG protein into vitellogenic females demonstrates that during the first 24 hr after injection, accumulation by the ovary is rapid. Immunoprecipitation and immunoelectrophoretic studies indicate that some of the ARG protein is accumulated unchanged. It is proposed that when the male transfers several spermatophores during copulation, he may make a significant contribution of protein to the developing oöcytes.     

Naphthoquinone-dependent generation of superoxide radicals by quinone reductase isolated from the plasma membrane of soybean

Using a tetrazolium-based assay, a NAD(P)H oxidoreductase was purified from plasma membranes prepared from soybean (Glycine max) hypocotyls. The enzyme, a tetramer of 85 kD, produces O2(.-) by a reaction that depended on menadione or several other 1,4-naphthoquinones, in apparent agreement with a classification as a one-electron-transferring flavoenzyme producing semiquinone radicals. However, the enzyme displayed catalytic and molecular properties of obligatory two-electron-transferring quinone reductases of the DT-diaphorase type, including insensitivity to inhibition by diphenyleneiodonium. This apparent discrepancy was clarified by investigating the pH-dependent reactivity of menadionehydroquinone toward O2 and identifying the protein by mass spectrometry and immunological techniques. The enzyme turned out to be a classical NAD(P)H:quinone-acceptor oxidoreductase (EC 1.6.5.2, formerly 1.6.99.2) that reduces menadione to menadionehydroquinone and subsequently undergoes autoxidation at pH > or = 6.5. Autoxidation involves the production of the semiquinone as an intermediate, creating the conditions for one-electron reduction of O2. The possible function of this enzyme in the generation of O2(.-) and H2O2 at the plasma membrane of plants in vivo is discussed.     

YidC Occupies the Lateral Gate of the SecYEG Translocon and Is Sequentially Displaced by a Nascent Membrane Protein

Most membrane proteins are co-translationally inserted into the lipid bilayer via the universally conserved SecY complex and they access the lipid phase presumably via a lateral gate in SecY. In bacteria, the lipid transfer of membrane proteins from the SecY channel is assisted by the SecY-associated protein YidC, but details on the SecY-YidC interaction are unknown. By employing an in vivo and in vitro site-directed cross-linking approach, we have mapped the SecY-YidC interface and found YidC in contact with all four transmembrane domains of the lateral gate. This interaction did not require the SecDFYajC complex and was not influenced by SecA binding to SecY. In contrast, ribosomes dissociated the YidC contacts to lateral gate helices 2b and 8. The major contact between YidC and the lateral gate was lost in the presence of ribosome nascent chains and new SecY-YidC contacts appeared. These data demonstrate that the SecY-YidC interaction is influenced by nascent-membrane-induced lateral gate movements.     

Indicators for the on-farm assessment of crop cultivar and livestock breed diversity: a survey-based participatory approach

Agrobiodiversity plays a fundamental role in guaranteeing food security. However, still little is known about the diversity within crop and livestock species: the genetic diversity. In this paper we present a set of indicators of crop accession and breed diversity for different farm types at farm-level, which may potentially supply a useful tool to assess and monitor farming system agrobiodiversity in a feasible and relatively affordable way. A generic questionnaire was developed to capture the information on crops and livestock in 12 European case study regions and in Uganda by 203 on-farm interviews. Through a participatory approach, which involved a number of stakeholders, eight potential indicators were selected and tested. Five of them are recommended as potentially useful indicators for agrobiodiversity monitoring per farm: (1) crop-species richness (up to 16 crop species), (2) crop-cultivar diversity (up to 15 crop cultivars, 1–2 on average), (3) type of crop accessions (landraces accounted for 3 % of all crop cultivars in Europe, 31 % in Uganda), (4) livestock-species diversity (up to 5 livestock species), and (5) breed diversity (up to five cattle and eight sheep breeds, on average 1–2).We demonstrated that the selected indicators are able to detect differences between farms, regions and dominant farm types. Given the present rate of agrobiodiversity loss and the dramatic effects that this may have on food production and food security, extensive monitoring is urgent. A consistent survey of crop cultivars and livestock breeds on-farm will detect losses and help to improve strategies for the management and conservation of on-farm genetic resources.     

The luminal helix l of PsaB is essential for recognition of plastocyanin or cytochrome c6 and fast electron transfer to photosystem I in Chlamydomonas reinhardtii.

At the lumenal side of photosystem I (PSI) in cyanobacteria, algae, and vascular plants, proper recognition and binding of the donor proteins plastocyanin (pc) and cytochrome (cyt) c(6) are crucial to allow subsequent efficient electron transfer to the photooxidized primary donor. To characterize the surface regions of PSI needed for the correct binding of both donors, loop j of PsaB of Chlamydomonas reinhardtii was modified using site-directed mutagenesis and chloroplast transformation. Mutant strains D624K, E613K/D624K, E613K/W627F, and D624K/W627F accumulated <20% of PSI as compared with wild type and were only able to grow photoautotrophically at low light intensities. Mutant strains E613N, E613K, and W627F accumulated >50% of PSI as compared with wild type. This was sufficient to isolate the altered PSI and perform a detailed analysis of the electron transfer between the modified PSI and the two algal donors using flash-induced spectroscopy. Such an analysis indicated that residue Glu(613) of PsaB has two functions: (i) it is crucial for an improved unbinding of the two donors from PSI, and (ii) it orientates the positively charged N-terminal domain of PsaF in a way that allows efficient binding of pc or cyt c(6) to PSI. Mutation of Trp(627) to Phe completely abolishes the formation of an intermolecular electron transfer complex between pc and PSI and also drastically diminishes the rate of electron transfer between the donor and PSI. This mutation also hinders binding and electron transfer between the altered PSI and cyt c(6). It causes a 10-fold increase of the half-time of electron transfer within the intermolecular complex of cyt c(6) and PSI. These data strongly suggest that Trp(627) is a key residue of the recognition site formed by the core of PSI for binding and electron transfer between the two soluble electron donors and the photosystem.