Species-specific differences of the spectroscopic properties of P700: analysis of the influence of non-conserved amino acid residues by site-directed mutagenesis of photosystem I from Chlamydomonas reinhardtii

We applied optical spectroscopy, magnetic resonance techniques, and redox titrations to investigate the properties of the primary electron donor P700 in photosystem I (PS I) core complexes from cyanobacteria (Thermosynechococcus elongatus, Spirulina platensis, and Synechocystis sp. PCC 6803), algae (Chlamydomonas reinhardtii CC2696), and higher plants (Spinacia oleracea). Remarkable species-specific differences of the optical properties of P700 were revealed monitoring the (3P700-P700) and (P700+.-P700) absorbance and CD difference spectra. The main bleaching band in the Qy region differs in peak position and line width for the various species. In cyanobacteria the absorbance of P700 extends more to the red compared with algae and higher plants which is favorable for energy transfer from red core antenna chlorophylls to P700 in cyanobacteria. The amino acids in the environment of P700 are highly conserved with two distinct deviations. In C. reinhardtii a Tyr is found at position PsaB659 instead of a Trp present in all other organisms, whereas in Synechocystis a Phe is found instead of a Trp at the homologous position PsaA679. We constructed several mutants in C. reinhardtii CC2696. Strikingly, no PS I could be detected in the mutant YW B659 indicating steric constraints unique to this organism. In the mutants WA A679 and YA B659 significant changes of the spectral features in the (3P700-P700), the (P700+.-P700) absorbance difference and in the (P700+.-P700) CD difference spectra are induced. The results indicate structural differences among PS I from higher plants, algae, and cyanobacteria and give further insight into specific protein-cofactor interactions contributing to the optical spectra.     

Conformational relaxation following reduction of the photoactive bacteriopheophytin in reaction centers from Balstochloris viridis. Influence of mutations at position M208

The photochemically trapped bacteriopheophytin (BPh) b radical anion in the active branch (phi(*-)A) of reaction centers (RCs) from Blastochloris (formerly called Rhodopseudomonas) viridis is characterized by 1H-ENDOR as well as optical absorption spectroscopy. The two site-directed mutants YF(M208) and YL(M208), in which tyrosine at position M208 is replaced by phenylalanine and leucine, respectively, are investigated and compared with the wild type. The residue at M208 is in close proximity to the primary electron donor, P, the monomeric bacteriochlorophyll (BCh1), B(A), and the BPh, phiA, that are involved in the transmembrane electron transfer to the quinone, Q(A), in the RC. The analysis of the ENDOR spectra of (phi(*-)A at 160 K indicates that two distinct states of phi(*-)A are present in the wild type and the mutant YF(M208). Based on a comparison with phi(*-)A in RCs of Rhodobacter sphaeroides the two states are interpreted as torsional isomers of the 3-acetyl group of phiA. Only one phi(*-)A state occurs in the mutant YL(M208). This effect of the leucine residue at position M208 is explained by steric hindrance that locks the acetyl group in one specific position. On the basis of these results, an interpretation of the optical absorption difference spectrum of the state phi(*-)AQ(*-)A is attempted. This state can be accumulated at 100 K and undergoes an irreversible change between 100 and 200 K [Tiede et al., Biochim. Biophys. Acta 892 (1987) 294-302]. The corresponding absorbance changes in the BCh1 Q(x) and Q(y) regions observed in the wild type also occur in the YF(M208) mutant but not in YL(M208). The observed changes in the wild type and YF(M208) are assigned to RCs in which the 3-acetyl group of phiA changes its orientation. It is concluded that this distinct structural relaxation of phiA can significantly affect the optical properties of B(A) and contribute to the light-induced absorption difference spectra.     

Detection of heme oxygenase activity in a library of four-helix bundle proteins: towards the de novo synthesis of functional heme proteins

Design and chemical synthesis of de novo heme proteins with enzymatic activity on cellulose membranes is described. 352 antiparallel four-helix bundle proteins with a single histidine for heme ligation were assembled from three different sets of short amphipathic helices on membrane-bound peptide templates. The templates were coupled by linkers to cellulose membranes of microplate format, which could be cleaved for control of intermediate and final products. The incorporation of heme and the heme oxygenase activity of the 352 proteins were monitored by measuring UV-visible spectra directly on the cellulose. The kinetics of the heme oxygenase reaction was studied by monitoring the decrease of the Soret band and the transient absorbance of verdoheme being an intermediate product in the formation of biliverdin. Four of the proteins covering a broad range of the enzymatic rate constants were selected and synthesized in solution for further characterization. Detailed studies by redox potentiometry, analytical ultracentrifugation, and electron paramagnetic resonance spectroscopy yielded information about the aggregation state of the proteins, the spin state and the putative coordination environment of the iron. The amount of five-coordinated high-spin iron and a positive reduction potential were found to promote the oxygenase activity of the proteins.     

Primary donor triplet states of Photosystem I and II studied by Q-band pulse ENDOR spectroscopy

Photosynthesis Research - The photoexcited triplet state of the “primary donors” in the two photosystems of oxygenic photosynthesis has been investigated by means of electron-nuclear...     

Characterization of a cyanobacterial-like uptake [NiFe] hydrogenase: EPR and FTIR spectroscopic studies of the enzyme from Acidithiobacillus ferrooxidans

Electron paramagnetic resonance (EPR) and Fourier transform IR studies on the soluble hydrogenase from Acidithiobacillus ferrooxidans are presented. In addition, detailed sequence analyses of the two subunits of the enzyme have been performed. They show that the enzyme belongs to a group of uptake [NiFe] hydrogenases typical for Cyanobacteria. The sequences have also a close relationship to those of the H₂-sensor proteins, but clearly differ from those of standard [NiFe] hydrogenases. It is concluded that the structure of the catalytic centre is similar, but not identical, to that of known [NiFe] hydrogenases. The active site in the majority of oxidized enzyme molecules, 97% in cells and more than 50% in the purified enzyme, is EPR-silent. Upon contact with H₂ these sites remain EPR-silent and show only a limited IR response. Oxidized enzyme molecules with an EPR-detectable active site show a Ni_r*-like EPR signal which is light-sensitive at cryogenic temperatures. This is a novelty in the field of [NiFe] hydrogenases. Reaction with H₂ converts these active sites to the well-known Ni_a-C* state. Illumination below 160 K transforms this state into the Ni_a-L* state. The reversal, in the dark at 200 K, proceeds via an intermediate Ni EPR signal only observed with the H₂-sensor protein from Ralstonia eutropha. The EPR-silent active sites in as-isolated and H₂-treated enzyme are also light-sensitive as observed by IR spectra at cryogenic temperatures. The possible origin of the light sensitivity is discussed. This study represents the first spectral characterization of an enzyme of the group of cyanobacterial uptake hydrogenases. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

The unusal redox centers of SoxXA, a novel c-type heme-enzyme essential for chemotrophic sulfur-oxidation of Paracoccus pantotrophus

The heterodimeric hemoprotein SoxXA, essential for lithotrophic sulfur oxidation of the aerobic bacterium Paracoccus pantotrophus, was examined by a combination of spectroelectrochemistry and EPR spectroscopy. The EPR spectra for SoxXA showed contributions from three paramagnetic heme iron centers. One highly anisotropic low-spin (HALS) species (gmax = 3.45) and two "standard" cytochrome-like low-spin heme species with closely spaced g-tensor values were identified, LS1 (gz = 2.54, gy = 2.30, and gx = 1.87) and LS2 (gz = 2.43, gy = 2.26, and gx = 1.90). The crystal structure of SoxXA from P. pantotrophus confirmed the presence of three heme groups, one of which (heme 3) has a His/Met axial coordination and is located on the SoxX subunit [Dambe et al. (2005) J. Struct. Biol. 152, 229-234]. This heme was assigned to the HALS species in the EPR spectra of the isolated SoxX subunit. The LS1 and LS2 species were associated with heme 1 and heme 2 located on the SoxA subunit, both of which have EPR parameters characteristic for an axial His/thiolate coordination. Using thin-layer spectroelectrochemistry the midpoint potentials of heme 3 and heme 2 were determined: Em3 = +189 +/- 15 mV and Em2 = -432 +/- 15 mV (vs NHE, pH 7.0). Heme 1 was not reducible even with 20 mM titanium(III) citrate. The Em2 midpoint potential turned out to be pH dependent. It is proposed that heme 2 participates in the catalysis and that the cysteine persulfide ligation leads to the unusually low redox potential (-436 mV). The pH dependence of its redox potential may be due to (de)protonation of the Arg247 residue located in the active site.     

Phi X174 protein E-mediated lysis of Escherichia coli

Bacteriophage PhiX174 encodes a single lysis gene, E, the function of which is necessary and sufficient to induce lysis of Escherichia coli. Here we present a novel model for E-lysis: physiological, genetic and biochemical data are presented which suggest that a transmembrane tunnel penetrating the inner and outer membrane is formed during the lytic action of protein E. Moreover, using high magnification scanning and transmission electron microscopy in this study, it was possible to visualize the transmembrane lysis structure directly.     

Dynamics of PhiX174 protein E-mediated lysis of Escherichia coli

Expression of cloned gene E of bacteriophage PhiX174 induces lysis by formation of a transmembrane tunnel structure in the cell envelope of Escherichia coli. Ultrastructural studies of the location of the lysis tunnel indicate that it is preferentially located at the septum or at polar regions of the cell. Furthermore, the diameter and shape of individual tunnel structures vary greatly indicating that its structure is not rigid. Apparently, the contours of individual lysis tunnels are determined by enlarged meshes in the peptidoglycan net and the force produced at its orifice, by the outflow of cytoplasmic content. Once the tunnel is formed the driving force for the lysis process is the osmotic pressure difference between cytoplasm and medium. During the lysis process areas of the cytoplasmic membrane which are not tightly attached to the envelope are extended inward by the negative pressure produced during lysis. After cell lysis external medium can diffuse through the lysis tunnel filling the inner cell space of the still rigid bacterial ghosts.     

The role of context in elucidating drivers of animal movement

Despite its consequences for ecological processes and population dynamics, intra‐specific variability is frequently overlooked in animal movement studies. Consequently, the necessary resolution to reveal drivers of individual movement decisions is often lost as animal movement data are aggregated to infer average or population patterns. Thus, an empirical understanding of why a given movement pattern occurs remains patchy for many taxa, especially in marine systems. Nonetheless, movement is often rationalized as being driven by basic life history requirements, such as acquiring energy (feeding), reproduction, predator‐avoidance, and remaining in suitable environmental conditions. However, these life history requirements are central to every individual within a species and thus do not sufficiently account for the high intra‐specific variability in movement behavior and hence fail to fully explain the occurrence of multiple movement strategies within a species. Animal movement appears highly context dependent as, for example, within the same location, the behavior of both resident and migratory individuals is driven by life history requirements, such as feeding or reproduction, however different movement strategies are utilized to fulfill them. A systematic taxa‐wide approach that, instead of averaging population patterns, incorporates and utilizes intra‐specific variability to enable predictions as to which movement patterns can be expected under a certain context, is needed. Here, we use intra‐specific variability in elasmobranchs as a case study to introduce a stepwise approach for studying animal movement drivers that is based on a context‐dependence framework. We examine relevant literature to illustrate how this context‐focused approach can aid in reliably identifying drivers of a specific movement pattern. Ultimately, incorporating behavioral variability in the study of movement drivers can assist in making predictions about behavioral responses to environmental change, overcoming tagging biases, and establishing more efficient conservation measures.     

The iron-oxygen reconstitution reaction in protein R2-Tyr-177 mutants of mouse ribonucleotide reductase. Epr and electron nuclear double resonance studies on a new transient tryptophan radical.

The ferrous iron/oxygen reconstitution reaction in protein R2 of mouse and Escherichia coli ribonucleotide reductase (RNR) leads to the formation of a stable protein-linked tyrosyl radical and a mu-oxo-bridged diferric iron center, both necessary for enzyme activity. We have studied the reconstitution reaction in three protein R2 mutants Y177W, Y177F, and Y177C of mouse RNR to investigate if other residues at the site of the radical forming Tyr-177 can harbor free radicals. In Y177W we observed for the first time the formation of a tryptophan radical in protein R2 of mouse RNR with a lifetime of several minutes at room temperature. We assign it to an oxidized neutral tryptophan radical on Trp-177, based on selective deuteration and EPR and electron nuclear double resonance spectroscopy in H2O and D2O solution. The reconstitution reaction at 22 degrees C in both Y177F and Y177C leads to the formation of a so-called intermediate X which has previously been assigned to an oxo (hydroxo)-bridged Fe(III)/Fe(IV) cluster. Surprisingly, in both mutants that do not have successor radicals as Trp. in Y177W, this cluster exists on a much longer time scale (several seconds) at room temperature than has been reported for X in E. coli Y122F or native mouse protein R2. All three mouse R2 mutants were enzymatically inactive, indicating that only a tyrosyl radical at position 177 has the capability to take part in the reduction of substrates.