Iris von der Hocht

Translational Diffusion and Interaction of a Photoreceptor and Its Cognate Transducer Observed in Giant Unilamellar Vesicles by Using Dual-Focus FCS

In order to monitor membrane–protein binding in lipid bilayers at physiological protein concentrations, we employed the recently developed dual‐focus fluorescence correlation spectroscopy (2fFCS) technique. In a case study on a photoreceptor consisting of seven transmembrane helices and its cognate transducer (two transmembrane helices), the lateral diffusion for these integral membrane proteins was analyzed in giant unilamellar vesicles (GUVs). The two‐dimensional diffusion coefficients of both separately diffusing proteins differ significantly, with D=2.2×10−8 cm2 s−1 for the photoreceptor and with D=4.1×10−8 cm2 s−1 for the transducer. In GUVs with both membrane proteins present together, we observed significantly smaller diffusion coefficients for labelled transducer molecules; this indicates the presence of larger diffusing units and therefore intermolecular protein binding. Based on the phenomenological dependence of diffusion coefficients on the molecules cylindrical radius, we are able to estimate the degree of membrane protein binding on a quantitative level.

Evidence for distinct electron transfer processes in terminal oxidases from different origin by means of protein film voltammetry.

Cytochrome aa3 from Paracoccus denitrificans and cytochrome ba3 from Thermus thermophilus, two distinct members of the heme–copper oxidase superfamily, were immobilized on electrodes modified with gold nanoparticles. This procedure allowed us to achieve direct electron transfer between the enzyme and the gold nanoparticles and to obtain evidence for different electrocatalytic properties of the two enzymes. The pH dependence and thermostability reveal that the enzymes are highly adapted to their native environments. These results suggest that evolution resulted in different solutions to the common problem of electron transfer to oxygen.

True wild type and recombinant wild type cytochrome c oxidase from Paracoccus denitrificans show a 20-fold difference in their catalase activity

The four subunit (SU) aa(3) cytochrome c oxidase (CcO) from Paracoccus denitrificans is one of the terminal enzymes of the respiratory chain. Its binuclear active center, residing in SU I, contains heme a(3) and Cu(B). Apart from its oxygen reductase activity, the protein possesses a peroxidase and a catalase activity. To compare variants and the wild type (WT) protein in a more stringent way, a recombinant (rec.) WT strain was constructed, carrying the gene for SU I on a low copy number plasmid. This rec. WT showed no difference in oxygen reductase activity compared to the American Type Culture Collection (ATCC) WT CcO but surprisingly its catalase activity was increased by a factor of 20. The potential over-production of SU I might impair the correct insertion of heme a(3) and Cu(B) because of a deficiency in metal inserting chaperones. An altered distance between heme a(3) and Cu(B) and variations in protein structure are possible reasons for the observed increased catalase activity. The availability of chaperones was improved by cloning the genes ctaG and surf1c on the same plasmid as the SU I gene. The new rec. WT CcO showed in fact a reduced catalase activity. Using differential scanning calorimetry no significant difference in thermal stability between the ATCC WT CcO and the rec. WT CcO was detected. However, upon aging the thermal stability of the rec. WT CcO was reduced compared to that of the ATCC WT CcO pointing to a decreased structural stability of the rec. WT CcO.

Measuring large numerical apertures by imaging the angular distribution of radiation of fluorescing molecules

Exact knowledge of the numerical aperture is crucial in many applications using high-aperture objectives such as confocal microscopy, optical trapping, or advanced sub-wavelength imaging methods. We propose and apply a precise and straightforward method for measuring this fundamental parameter of microscope objectives with numerical apertures above unity. Our method exploits the peculiarities of the fluorescence emission of molecules at a glass/air interface.

Resonance Raman Characterization of the Ammonia-Generated Oxo Intermediate of Cytochrome c Oxidase from Paracoccus denitrificans

A novel oxo state of cytochrome c oxidase from Paracoccus denitrificans generated by successive addition of excess H2O2 and ammonia was investigated using resonance Raman (RR) spectroscopy. Addition of ammonia to the H2O2-generated artificial F state resulted in an upshift of the oxoferryl stretching vibration from 790 to 796 cm(-1), indicating that ammonia influences ligation of the heme-bound oxygen in the binuclear center. Concomitantly performed RR measurements in the high-frequency region between 1300 and 1700 cm(-1) showed a high-spin to low-spin transition of heme a3 upon generation of the F state that was not altered by addition of ammonia. Removal of H2O2 by addition of catalase resulted in the disappearance of the oxoferryl stretching vibration and major back transformation of heme a3 into the high-spin state. The ratio of high-spin to low-spin states was identical for intermediates created with and without ammonia, leading to the conclusion that ammonia does not interact directly with heme a3. Only for the ammonia-created state was a band at 612 nm observed in the UV-visible difference spectrum that was shifted to 608 nm after addition of catalase. Our results support the hypothesis by von der Hocht et al. [von der Hocht, I., et al. (2011) Proc. Natl. Acad. Sci. U.S.A. 108, 3964-3969] that addition of ammonia creates a novel oxo intermediate state called PN where ammonia binds to CuB once the oxo intermediate F state has been formed.

Measuring precise diffusion coefficients with two-focus fluorescence correlation spectroscopy

We present a new method for precisely measuring diffusion coefficients of fluorescent molecules at nanomolar concentrations. The method is based on a modified Fluorescence Correlation Spectroscopy (FCS)-setup which is robust against many artifacts that are inherent to standard FCS 1, 2. The core idea of the new method is the introduction of an external ruler by generating two laterally shifted and overlapping laser foci at a fixed and known distance. Data fitting is facilitated by ab initio calculations of resulting correlation curves and subsequent affine transformation of these curves to match the measured auto- and cross-correlation functions. The affine transformation coefficient along the time axis then directly yields the correct diffusion coefficient. This method is not relying on the rather inexact assumption of a 3D Gaussian shaped detection volume. We measured the diffusion coefficient of the red fluorescent dye Atto-655 (Atto-Tec GmbH) in water and compared the obtained value with results from Gradient Pulsed Field NMR (GPF-NMR).

Interconversions of P and F intermediates of cytochrome c oxidase from Paracoccus denitrificans

Cytochrome c Oxidase (CcO) is the terminal enzyme of the respiratory chain. The redox driven proton pump catalyses the four electron reduction of molecular oxygen to water. Electrons are delivered by cytochrome c to the bimetallic CuA centre and transferred via haem a into the binuclear haem a3-CuB centre where the reduction of oxygen takes place. Elucidation of the intermediate structures in the catalytic cycle is crucial for understanding the mechanism of oxygen reduction. P and F states are doubly and triply reduced catalytic intermediates, respectively, when starting from oxidised CcO (O state). A P state can also be formed artificially by reaction of CcO with carbon monoxide or upon addition of equimolar amounts of hydrogen peroxide. Artificial intermediates are not necessarily the same as physiological intermediates but nevertheless one can learn what kind of reactions CcO can undergo. Here we show that the F state, classically generated by reaction with an excess of H2O2, can be converted into a new P state by addition of ammonia at pH 9. This new P state has a difference absorption maximum at 612 nm. Electron paramagnetic resonance experiments show that this new P state possesses an amino acid radical. Binding of ammonia to CcO is reversible upon lowering pH. Activity of CcO is fully maintained in the presence of ammonia. Assuming that ammonia coordinates to CuB, these results suggest that spectroscopic differences between P and F states are caused by different CuB ligands.

Monitoring of small conformational changes by high-precision measurements of hydrodynamic radius with 2-focus fluorescence correlation spectroscopy (2fFCS)

We report on our application of a new fluorescence-correlation spectroscopy technique, 2-focus FCS, for measuring the hydrodynamic radius of molecules with sub- Ångstrøm precision. The method is applied of monitoring conformational changes of proteins upon ion binding. In particular, we present measurements on Ca2+-binding of recoverin. Recoverin belongs to the superfamily of EF-hand Ca2+-binding proteins and operates as a Ca2+-sensor in vertebrate photoreceptor cells, where it regulates the activity of rhodopsin kinase GRK1 in a Ca2+-dependent manner. The protein undergoes conformational changes upon Ca2+-changes that are reflected as changes in their hydrodynamic radius. By using 2fFCS we were able to resolve hydrodynamic radius changes of ca. one Ångstrøm and used the Ca2+ dependence of this radius for recording binding curves in solution. We compare our results with those obtained by other techniques.