One at a Time: Intramolecular Electron-Transfer Kinetics in Small Laccase Observed during Turnover

Single-molecule enzymology provides an unprecedented level of detail about aspects of enzyme mechanisms which have been very difficult to probe in bulk. One such aspect is intramolecular electron transfer (ET), which is a recurring theme in the research on oxidoreductases containing multiple redox-active sites. We measure the intramolecular ET rates between the copper centers of the small laccase from <i>Streptomyces coelicolor</i> at room temperature and pH 7.4, one molecule at a time, during turnover. The forward and backward rates across many molecules follow a log-normal distribution with means of 460 and 85 s^–1, respectively, corresponding to activation energies of 347 and 390 meV for the forward and backward rates. The driving force and the reorganization energy amount to 0.043 and 1.5 eV, respectively. The spread in rates corresponds to a spread of ∼30 meV in the activation energy. The second-order rate constant for reduction of the T1 site amounts to 2.9 × 10⁴ M^–1 s^–1. The mean of the distribution of forward ET rates is higher than the turnover rate from ensemble steady-state measurements and, thus, is not rate limiting

Selected analysis of the fluorescence profiles across the edge of the bleached area in partially-bleached cells, allowing the real-time exploration of the fluorescence dynamics of PBsomes correlated to their lateral diffusion.

<p>A, untreated cell; B, betaine-treated cell; C, glutaraldehyde-treated cell. Area (1), the unbleached cellular area; Area (2), the slope of the fluorescence intensity gradient; Area (3), the fluorescence recovery as a function of distance from the edge of the bleached area. The mean fluorescence intensities of three different regions (R1, R2 and R3 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005295#pone-0005295-g004" target="_blank">Figure 4A–C</a>) at distinct recovery time were analyzed: D, from untreated cell; E, from betaine-treated cell; F, from glutaraldehyde-treated cell. Fluorescence intensities are normalized at zero second. As shown, no remarkable difference of the fluorescence intensities in these three cellular areas could be observed, indicating the fluorescence recovery of PBsomes does not differ as a function of distance from the edge of the bleached area of theses cells.</p

FRAP selected fluorescence images of <i>P. cruentum</i> WT cells pretreated with glutaraldehyde/betaine.

<p>Excitation is at 568 nm and detection range is from 650 to 750 nm. A, partially-bleached cell pretreated with betaine; B, wholly-bleached cell pretreated with betaine; C, partially-bleached cell pretreated with glutaraldehyde; D, wholly-bleached cell pretreated with glutaraldehyde. Scale bar: 5 µm.</p

FRAP selected fluorescence images of the <i>P. cruentum</i> WT cells.

<p>A, wholly bleaching on the cells. Excitation is at 568 nm and detection range is from 650 to 750 nm. Selected fluorescence images from sequences recorded before bleaching, immediately after bleaching of PBsomes, and at various time lapses. Cycle 1: 1-2-3; cycle 2: 3-4-5; cycle 3: 5-6-7; Scale bar: 5 µm; B, one-dimensional bleaching profiles derived from the sequences of fluorescence images of panel A.</p

Selected PE fluorescence images in cells imaged with confocal microscopy by detecting fluorescence at 550–600 nm. A, B, native WT cells; C, WT cell pretreated with glutaraldehyde; D, F11 cell. Scale bar: 5 µm.

<p>Selected PE fluorescence images in cells imaged with confocal microscopy by detecting fluorescence at 550–600 nm. A, B, native WT cells; C, WT cell pretreated with glutaraldehyde; D, F11 cell. Scale bar: 5 µm.</p

Qualitative FRAP experiments of red alga <i>P. cruentum</i> WT cell.

<p>A, selected fluorescence images from typical sequences recorded before bleaching, immediately after bleaching of PBsomes, and at various time lapses. Excitation is at 568 nm and detection range is from 650 to 750 nm. Scale bar: 5 µm; B, total fluorescence intensity of bleached cell region as a function of time. The recovery of the fluorescence is presented as square spots and fitted to an exponential function (solid line).</p

Super-resolution images of internalized α-syn aggregates in endosomal vesicles in time.

<p>(a) dSTORM image of a cell treated for half an hour with α-syn -Alexa532 aggregates. A detailed view of the aggregates in the cell membrane is shown below a). (b) After 2 hours of incubation, α-syn aggregates are internalized in vesicles. Detailed view of the aggregates in a vesicle shown in the image below b). (c) Internalized α-syn aggregates after 24 hours of incubation, with two different sized clusters highlighted bellow image c).</p

Characteristic properties of the optical setup.

<p>(a) Frame with the signal of several Alexa532 molecules. Scale bar = 2μm. (b) Histogram of the sigma of positional accuracy (Mean: 11 nm). (c) Zoom-in of the white square in Fig 1A showing the Gaussian intensity profile. (d) Histogram of the intensity of localizations (Mean: 447 photons).</p

Size distribution of α-syn aggregates in endosomal vesicles in time.

<p>(a)-(c) Histogram of FWHM of intracellular α-syn clusters in time. (ANOVA significance levels: (a)-(b): 10⁻⁴; (b)-(c):5×10⁻³; ((a)-(c):10⁻⁷). (d) A decrease in α-syn cluster size is observed in the mean average FWHM of α-syn clusters in time (median and 50% interval).</p

Internalization of α-syn sonicated fibrils in human neuroblastoma cells.

<p>Images show co-localization of Alexa 532 labeled α-syn aggregates (green) with LysoTracker Deep Red (red). SH-SY5Y cells were treated with 50 nM LysoTracker Deep Red, then washed, incubated further with Alexa532-labeled α-syn sonicated fibrils and imaged live on a confocal microscope.</p