Marc Brecht

A single-crystal ENDOR and density functional theory study of the oxidized states of the [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F.

The catalytic center of the [NiFe] hydrogenase of Desulfovibrio vulgaris Miyazaki F in the oxidized states was investigated by electron paramagnetic resonance and electron–nuclear double resonance spectroscopy applied to single crystals of the enzyme. The experimental results were compared with density functional theory (DFT) calculations. For the Ni-B state, three hyperfine tensors could be determined. Two tensors have large isotropic hyperfine coupling constants and are assigned to the β-CH2 protons of the Cys-549 that provides one of the bridging sulfur ligands between Ni and Fe in the active center. From a comparison of the orientation of the third hyperfine tensor with the tensor obtained from DFT calculations an OH− bridging ligand has been identified in the Ni-B state. For the Ni-A state broader signals were observed. The signals of the third proton, as observed for the “ready” state Ni-B, were not observed at the same spectral position for Ni-A, confirming a structural difference involving the bridging ligand in the “unready” state of the enzyme.

Protein dynamics-induced variation of excitation energy transfer pathways

Strong anticorrelation between the fluorescence emission of different emitters is observed by employing single-molecule fluorescence spectroscopy on photosystem I at cryogenic temperatures. This anticorrelation demonstrates a time-dependent interaction between pigments participating in the exciton transfer chain, implying that uniquely defined energy transfer pathways within the complex do not exist. Fluctuations of the chromophores themselves or their immediate protein surroundings induce changes in their site energy, and, as a consequence, these fluctuations change the coupling within the excitation transfer pathways. The time scales of the site energy fluctuations of the individual emitters do not meet the time scales of the observed correlated emission behavior. Therefore, the emitters must be fed individually by energetically higher lying states, causing the observed intensity variations. This phenomenon is shown for photosystem I pigment–protein complexes from 2 different cyanobacteria (Thermosynechococcus elongatus and Synechocystis sp. PCC 6803) with strongly different spectral properties underlining the general character of the findings. The variability of energy transfer pathways might play a key role in the extreme robustness of light-harvesting systems in general.

Spectral Diffusion Induced by Proton Dynamics in Pigment-Protein Complexes

The fluorescence emission of individual photosystem I complexes from Synechocystis PCC 6803 in protonated and deuterated buffer shows zero-phonon lines as well as broad intensity distributions. The number and the line width of the zero phonon lines depend strongly on the solvent (H(2)O/D(2)O). The spectral diffusion rate of the whole fluorescence emission from photosystem I is significantly reduced upon deuteration of the solvent. This leads to a substantial increase of well-resolved zero-phonon lines. Since the chlorophyll a chromophores lack exchangeable protons, these observed changes in the spectral diffusion have to be assigned to exchangeable protons at the amino acids and structural water molecules in the chromophore binding pocket.

Fluorescence of the various red antenna states in photosystem I complexes from cyanobacteria is affected differently by the redox state of P700

Photosystem I of cyanobacteria contains different spectral pools of chlorophylls called red or long-wavelength chlorophylls that absorb at longer wavelengths than the primary electron donor P700. We measured the fluorescence spectra at the ensemble and the single-molecule level at low temperatures in the presence of oxidized and reduced P700. In accordance with the literature, it was observed that the fluorescence is quenched by P700(+). However, the efficiency of the fluorescence quenching by oxidized P700(+) was found to be extremely different for the various red states in PS I from different cyanobacteria. The emission of the longest-wavelength absorbing antenna state in PS I trimers from Thermosynechococcus elongatus (absorption maximum at 5K: ≈ 719nm; emission maximum at 5K: ≈ 740nm) was found to be strongly quenched by P700(+) similar to the reddest state in PS I trimers from Arthrospira platensis emitting at 760nm at 5K. The fluorescence of these red states is diminished by more than a factor of 10 in the presence of oxidized P700. For the first time, the emission of the reddest states in A. platensis and T. elongatus has been monitored using single-molecule fluorescence techniques.

The H(2) sensor of Ralstonia eutropha: biochemical and spectroscopic analysis of mutant proteins modified at a conserved glutamine residue close to the [NiFe] active site.

Abstract. [NiFe] hydrogenases contain a highly conserved histidine residue close to the [NiFe] active site which is altered by a glutamine residue in the H2-sensing [NiFe] hydrogenases. In this study, we exchanged the respective glutamine residue of the H2 sensor (RH) of Ralstoniaeutropha, Q67 of the RH large subunit HoxC, by histidine, asparagine and glutamate. The replacement by histidine and asparagine resulted in slightly unstable RH proteins which were hardly affected in their regulatory and enzymatic properties. The exchange to glutamate led to a completely unstable RH protein. The purified wild-type RH and the mutant protein with the Gln/His exchange were analysed by continuous-wave and pulsed electron paramagnetic resonance (EPR) techniques. We observed a coupling of a nitrogen nucleus with the [NiFe] active site for the mutant protein which was absent in the spectrum of the wild-type RH. A combination of theoretical calculations with the experimental data provided an explanation for the observed coupling. It is shown that the coupling is due to the formation of a weak hydrogen bond between the protonated N(ε) nucleus of the histidine with the sulfur of a conserved cysteine residue which coordinates the metal atoms of the [NiFe] active site as a bridging ligand. The effect of this hydrogen bond on the local structure of the [NiFe] active site is discussed.

Long-wavelength limit of photochemical energy conversion in Photosystem I.

In Photosystem I (PS I) long-wavelength chlorophylls (LWC) of the core antenna are known to extend the spectral region up to 750 nm for absorbance of light that drives photochemistry. Here we present clear evidence that even far-red light with wavelengths beyond 800 nm, clearly outside the LWC absorption bands, can still induce photochemical charge separation in PS I throughout the full temperature range from 295 to 5 K. At room temperature, the photoaccumulation of P700+• was followed by the absorbance increase at 826 nm. At low temperatures (T < 100 K), the formation of P700+•FA/B–• was monitored by the characteristic EPR signals of P700+• and FA/B–• and by the characteristic light-minus-dark absorbance difference spectrum in the QY region. P700 oxidation was observed upon selective excitation at 754, 785, and 808 nm, using monomeric and trimeric PS I core complexes of Thermosynechococcus elongatus and Arthrospira platensis, which differ in the amount of LWC. The results show that the LWC cannot be responsible for the long-wavelength excitation-induced charge separation at low temperatures, where thermal uphill energy transfer is frozen out. Direct energy conversion of the excitation energy from the LWC to the primary radical pair, e.g., via a superexchange mechanism, is excluded, because no dependence on the content of LWC was observed. Therefore, it is concluded that electron transfer through PS I is induced by direct excitation of a proposed charge transfer (CT) state in the reaction center. A direct signature of this CT state is seen in absorbance spectra of concentrated PS I samples, which reveal a weak and featureless absorbance band extending beyond 800 nm, in addition to the well-known bands of LWC (C708, C719 and C740) in the range between 700 and 750 nm. The present findings suggest that nature can exploit CT states for extending the long wavelength limit in PSI even beyond that of LWC. Similar mechanisms may work in other photosynthetic systems and in chemical systems capable of photoinduced electron transfer processes in general.

Red Antenna States of Photosystem I from Synechocystis PCC 6803

Single-molecule spectroscopy at low temperatures was used to elucidate spectral properties, heterogeneities, and dynamics of the red-shifted chlorophyll a (Chl a) molecules responsible for the fluorescence from photosystem I (PSI). Emission spectra of single PSI complexes from the cyanobacterium Synechocystis PCC 6803 show zero-phonon lines (ZPLs) as well as broad intensity distributions without ZPLs. ZPLs are found most frequently on the blue side of the broad intensity distributions. The abundance of ZPLs decreases almost linearly at longer wavelengths. The distribution of ZPLs indicates the existence of at least two pools with maxima at 699 and 710 nm. The pool with the maximum at 710 nm is assigned to chlorophylls absorbing around 706 nm (C706), whereas the pool with the maximum at 699 nm (F699) can be assigned to chlorophylls absorbing at 692, 695, or 699 nm. The broad distributions dominating the red side of the spectra are made up of a low number of emitters assigned to the red-most pool C714. The properties of F699 show close relation to those of F698 in Synechococcus PCC 7002 and C708 in Thermosynechococcus elongatus. Furthermore, a high similarity is found between the C714 pool in Synechocystis PCC 6803 and C708 in Synechococcus PCC 7002 as well as C719 in T. elongatus.

Effect of Glycerol and PVA on the Conformation of Photosystem I

Single-molecule spectroscopy at cryogenic temperatures was used to examine the impact of buffer solution, glycerol/buffer mixtures (25% and 66%), and poly(vinyl alcohol) (PVA) films on the conformation of photosystem I (PSI) from Thermosynechoccocus elongatus. PSI holds a number of chromophores embedded at different places within the protein complex that show distinguishable fluorescence at low temperatures. The fluorescence emission from individual complexes shows inter- and intracomplex heterogeneity depending on the solution wherein PSI was dissolved. Statistical evaluation of spectra of a large number of complexes shows that the fluorescence emission of some of these chromophores can be used as sensors for their local nanoenvironment and some as probe for the conformation of the whole protein complex. Preparation in glycerol/buffer mixtures yields a high homogeneity for all chromophores, indicating a more compact protein conformation with less structural variability. In buffer solution a distinct heterogeneity of the chromophores is observed. PSI complexes in PVA show highly heterogeneous spectra as well as a remarkable blue shift of the fluorescence emission, indicating a destabilization of the protein complex. Photosystem I prepared in PVA cannot be considered fully functional, and conclusions drawn from experiments with PSI in PVA films are of questionable value.

Spectroscopic characterization of photosystem I at the single-molecule level

Progress in the field of single-molecule techniques has yielded enormous possibilities for studying photosynthetic pigment–protein complexes. The aim of this review is to present recent developments in spectroscopy on single photosystem I (PSI) complexes. The fluorescence emission of PSI is composed of the contributions of several chlorophyll a (Chl a) species, showing a remarkable red-shift compared with the fluorescence emission of Chl a in solution. Thus far, single complexes of PSI from Th. elongatus, Synechocystis PCC 6803, and Synechococcus PCC 7002 have been studied. Even on single-molecule level, several emitters contribute to the fluorescence emission of PSI. The spectral characteristics of these emitters found for the different organisms are compared with findings from ensemble and site-selective spectroscopy. Based on observed spectral positions and spectral diffusion properties, an assignment of different red emitters to specific positions in the X-ray structure is discussed. Favorable optical properties of PSI allow studies concerning the influence of solvent exchange on spectral diffusion. Remarkable changes in the spectral diffusion rate were observed upon H2O to D2O exchange. These results are discussed in the framework of pigment–protein coupling. Further, the fluorescence emission of different emitters within one single PSI complex shows anti-correlated intensity variations during time. This indicates changes in the excitation energy transfer pathways within a single PSI complex. Implication for excitation energy transfer within multi-chromophore systems are discussed.

Confocal sample-scanning microscope for single-molecule spectroscopy and microscopy with fast sample exchange at cryogenic temperatures.

The construction of a microscope with fast sample transfer system for single-molecule spectroscopy and microscopy at low temperatures using 2D/3D sample-scanning is reported. The presented construction enables the insertion of a sample from the outside (room temperature) into the cooled (4.2 K) cryostat within seconds. We describe the mechanical and optical design and present data from individual Photosystem I complexes. With the described setup numerous samples can be investigated within one cooling cycle. It opens the possibility to investigate biological samples (i) without artifacts introduced by prolonged cooling procedures and (ii) samples that require preparation steps like plunge-freezing or specific illumination procedures prior to the insertion into the cryostat.