Chemical Exchange at the Trinuclear Copper Center of Small Laccase from Streptomyces coelicolor.

The trinuclear copper center (TNC) of laccase reduces oxygen to water with very little overpotential. The arrangement of the coppers and ligands in the TNC is known to be from many crystal structures, yet information about possible dynamics of the ligands is absent. Here, we report dynamics at the TNC of small laccase from Streptomyces coelicolor using paramagnetic NMR and electron paramagnetic resonance spectroscopy. Fermi contact-shifted resonances tentatively assigned to histidine Hδ1 display a two-state chemical exchange with exchange rates in the order of 100 s-1. In the electron paramagnetic resonance spectra, at least two forms are observed with different gz-values. It is proposed that the exchange processes reflect the rotational motion of histidine imidazole rings that coordinate the coppers in the TNC.

Single electron transfer events and dynamical heterogeneity in the small protein azurin from Pseudomonas aeruginosa.

Monitoring the fluorescence of single-dye-labeled azurin molecules, we observed the reaction of azurin with hexacyanoferrate under controlled redox potential yielding data on the timing of individual (forward and backward) electron transfer (ET) events. Change-point analysis of the time traces demonstrates significant fluctuations of ET rates and of mid-point potential E 0. These fluctuations are a signature of dynamical heterogeneity, here observed on a 14 kDa protein, the smallest to date. By correlating changes in forward and backward reaction rates we found that 6% of the observed change events could be explained by a change in midpoint potential, while for 25% a change of the donor-acceptor coupling could explain the data. The remaining 69% are driven by variations in complex association constants or structural changes that cause forward and back ET rates to vary independently. Thus, the observed spread in individual ET rates could be related in a unique way to variations in molecular parameters. The relevance for the understanding of metabolic processes is briefly discussed.

Fluorescence Correlation Spectroscopy of Labeled Azurin Reveals Photoinduced Electron Transfer between Label and Cu Center.

Fluorescent labeling of biomacromolecules enjoys increasing popularity for structural, mechanistic, and microscopic investigations. Its success hinges on the ability of the dye to alternate between bright and dark states. Förster resonance energy transfer (FRET) is an important source of fluorescence modulation. Photo-induced electron transfer (PET) may occur as well, but is often considered only when donor and acceptor are in van der Waals contact. In this study, PET is shown between a label and redox centers in oxidoreductases, which may occur over large distances. In the small blue copper protein azurin, labeled with ATTO655, PET is observed when the label is at 18.5 Å, but not when it is at 29.1 Šfrom the Cu. For CuII , PET from label to Cu occurs at a rate of (4.8±0.3)×104  s-1 and back at (0.7±0.1)×103  s-1 . With CuI the numbers are (3.3±0.7)×106  s-1 and (1.0±0.1)×104  s-1 . Reorganization energies and electronic coupling elements are in the range of 0.8-1.2 eV and 0.02-0.5 cm-1 , respectively. These data are compatible with electron transfer (ET) along a through-bond pathway although transient complex formation followed by ET cannot be ruled out. The outcome of this study is a useful guideline for experimental designs in which oxidoreductases are labelled with fluorescent dyes, with particular attention to single molecule investigations. The labelling position for FRET can be optimized to avoid reactions like PET by evaluating the structure and thermodynamics of protein and label.

Direct Observation of α-Synuclein Amyloid Aggregates in Endocytic Vesicles of Neuroblastoma Cells.

Aggregation of α-synuclein has been linked to both familial and sporadic Parkinson's disease. Recent studies suggest that α-synuclein aggregates may spread from cell to cell and raise questions about the propagation of neurodegeneration. While continuous progress has been made characterizing α-synuclein aggregates in vitro, there is a lack of information regarding the structure of these species inside the cells. Here, we use confocal fluorescence microscopy in combination with direct stochastic optical reconstruction microscopy, dSTORM, to investigate α-synuclein uptake when added exogenously to SH-SY5Y neuroblastoma cells, and to probe in situ morphological features of α-synuclein aggregates with near nanometer resolution. We demonstrate that using dSTORM, it is possible to follow noninvasively the uptake of extracellularly added α-synuclein aggregates by the cells. Once the aggregates are internalized, they move through the endosomal pathway and accumulate in lysosomes to be degraded. Our dSTORM data show that α-synuclein aggregates remain assembled after internalization and they are shortened as they move through the endosomal pathway. No further aggregation was observed inside the lysosomes as speculated in the literature, nor in the cytoplasm of the cells. Our study thus highlights the super-resolution capability of dSTORM to follow directly the endocytotic uptake of extracellularly added amyloid aggregates and to probe the morphology of in situ protein aggregates even when they accumulate in small vesicular compartments.

Tracking electrons in biological macromolecules: from ensemble to single molecule.

Nature utilizes oxido-reductases to cater to the energy demands of most biochemical processes in respiratory species. Oxido-reductases are capable of meeting this challenge by utilizing redox active sites, often containing transition metal ions, which facilitate movement and relocation of electrons/protons to create a potential gradient that is used to energize redox reactions. There has been a consistent struggle by researchers to estimate the electron transfer rate constants in physiologically relevant processes. This review provides a brief background on the measurements of electron transfer rates in biological molecules, in particular Cu-containing enzymes, and highlights the recent advances in monitoring these electron transfer events at the single molecule level or better to say, at the individual event level.

Nanoscale methods for single-molecule electrochemistry.

The development of experiments capable of probing individual molecules has led to major breakthroughs in fields ranging from molecular electronics to biophysics, allowing direct tests of knowledge derived from macroscopic measurements and enabling new assays that probe population heterogeneities and internal molecular dynamics. Although still somewhat in their infancy, such methods are also being developed for probing molecular systems in solution using electrochemical transduction mechanisms. Here we outline the present status of this emerging field, concentrating in particular on optical methods, metal-molecule-metal junctions, and electrochemical nanofluidic devices.

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 Streptomyces coelicolor 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 × 10(4) 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.

Bi-enzyme sensor for phenolic compounds with fluorescent read-out.

In this paper, the use of tyrosinase (Ty) from Streptomyces antibioticus, labeled with a fluorescent tag, in combination with soluble quinoprotein (PQQ-containing) glucose dehydrogenase (s-GDH) to measure trace amounts of phenols is explored. Proof of concept is provided by a series of experiments, which show a clear quantitative dependence of the response on the phenol concentration. One of the advantages of the detection system is that apart from a standard fluorimeter no further instrumentation is required.

Involvement of Tyr108 in the enzyme mechanism of the small laccase from Streptomyces coelicolor.

The enzyme mechanism of the multicopper oxidase (MCO) SLAC from Streptomyces coelicolor was investigated by structural (XRD), spectroscopic (optical, EPR), and kinetics (stopped-flow) experiments on variants in which residue Tyr108 had been replaced by Phe or Ala through site-directed mutagenesis. Contrary to the more common three-domain MCOs, a tyrosine in the two-domain SLAC is found to participate in the enzyme mechanism by providing an electron during oxygen reduction, giving rise to the temporary appearance of a tyrosyl radical. The relatively low k(cat)/K(M) of SLAC and the involvement of Y108 in the enzyme mechanism may reflect an adaptation to a milieu in which there is an imbalance between the available reducing and oxidizing co-substrates. The purported evolutionary relationship between the two-domain MCOs and human ceruloplasmin appears to extend not only to the 3D structure and the mode of binding of the Cu's in the trinuclear center, as noted before, but also to the enzyme mechanism.

Probing redox proteins on a gold surface by single molecule fluorescence spectroscopy.

The interaction between the fluorescently labeled redox protein, azurin, and a thin gold film is characterized using single-molecule fluorescence intensity and lifetime measurements. Fluorescence quenching starts at distances below 2.3 nm from the gold surface. At shorter distances the quantum yield may decrease down to fourfold for direct attachment of the protein to bare gold. Outside of the quenching range, up to fivefold enhancement of the fluorescence is observed on average with increasing roughness of the gold layer. Fluorescence-detected redox activity of individual azurin molecules, with a lifetime switching ratio of 0.4, is demonstrated for the first time close to a gold surface.

Top-down FTICR MS for the identification of fluorescent labeling efficiency and specificity of the Cu-protein azurin.

Fluorescent protein labeling has been an indispensable tool in many applications of biochemical, biophysical, and cell biological research. Although detailed information about the labeling stoichiometry and exact location of the label is often not necessary, for other purposes, this information is crucial. We have studied the potential of top-down electrospray ionization (ESI)-15T Fourier transform ion cyclotron resonance (FTICR) mass spectrometry to study the degree and positioning of fluorescent labeling. For this purpose, we have labeled the Cu-protein azurin with the fluorescent label ATTO 655-N-hydroxysuccinimide(NHS)-ester and fractionated the sample using anion exchange chromatography. Subsequently, individual fractions were analyzed by ESI-15T FTICR to determine the labeling stoichiometry, followed by top-down MS fragmentation, to locate the position of the label. Results showed that, upon labeling with ATTO 655-NHS, multiple different species of either singly or doubly labeled azurin were formed. Top-down fragmentation of different species, either with or without the copper, resulted in a sequence coverage of approximately 50%. Different primary amine groups were found to be (potential) labeling sites, and Lys-122 was identified as the major labeling attachment site. In conclusion, we have demonstrated that anion exchange chromatography in combination with ultrahigh resolution 15T ESI-FTICR top-down mass spectrometry is a valuable tool for measuring fluorescent labeling efficiency and specificity.

Sensitive detection of histamine using fluorescently labeled oxido-reductases.

A detection scheme is described by which the histamine contents of biological samples can be established. The scheme is based on the use of methylamine dehydrogenase (MADH) which converts primary amines into the corresponding aldehydes and ammonia. The generated reducing equivalents are subsequently transferred to the physiological partner of MADH, amicyanin, which thereby is converted from the oxidized blue-colored form into the reduced colorless form. The change in absorption is detected by monitoring the fluorescence of a covalently attached Cy5 dye label whose fluorescence is (partly) quenched by Förster resonance energy transfer (FRET) to the Cu-site of the amicyanin. The quenching efficiency and, thereby, the label fluorescence, depends on the oxidation state of the amicyanin. When adding histamine to the assay mixture the proportionality between the substrate concentration and the observed rate of the fluorescence increase has enabled this assay as a sensor method with high sensitivity. The MADH and amicyanin composition can be tuned so that the sensor can be adapted over a broad range of histamine concentrations (13 nM-225 µM). The lowest concentration detected so far is 13 nM of histamine. The sensor retained its linearity up to 225 µM with a coefficient of variation of 11% for 10 measurements of 100nM histamine in a 100 µL sample volume. The use of a label fluorescing around 660 nm helps circumventing the interference from background fluorescence in biological samples. The sensor has been tested to detect histamine in biological fluids such as fish extracts and blood serum.

Orientational control over nitrite reductase on modified gold electrode and its effects on the interfacial electron transfer.

Recently, studies have been reported in which fluorescently labeled redox proteins have been studied with a combination of spectroscopy and electrochemistry. In order to understand the effect of the dye on the protein-electrode interaction, voltammetry and surface analysis have been performed on protein films of dye-labeled and unlabeled forms of a cysteine-surface variant (L93C) and the wild type (wt) of the copper containing nitrite reductase (NiR) from Alcaligenes faecalis S6. The protein has been adsorbed onto gold electrodes modified with self-assembled monolayers (SAMs) made up of 6-mercaptohexanol (6-OH) and mixtures of various octanethiols. Electrochemical and surface-analytical techniques were utilized to explore the influence of the SAM composition on wt and L93C NiR enzyme activity and the orientation of the enzyme molecules with respect to the electrode/SAM. The unlabeled L93C NiR enzyme is only electroactive on mixed SAMs composed of positive 8-aminooctanethiol (8-NH(2)) and 8-mercaptooctanol (8-OH). No enzymatic activity is observed on SAMs consisting of pure 6-OH, 8-OH, or pure 8-NH(2). Modification of L93C NiR with the ATTO 565 dye resulted in enzymatic activity on SAMs of 6-OH, but not on SAMs of 8-OH. Quartz crystal microbalance with dissipation measurements show that well-ordered and rigid protein films (single orientation of the protein) are formed when NiR is electroactive. By contrast, electrode-NiR combinations for which no electrochemical activity is observed still have NiR adsorbed on the surfaces, but a less-structured and water-rich film is formed. For the unlabeled L93C NiR, bilayer formation is observed, suggesting that the Cys93 residue is orientated away from the surface and able to form disulfide bridges to a second layer of L93C NiR. The results indicate that interfacial electron transfer is only possible if the negatively charged surface patch surrounding the electron-entry site of NiR is directed toward the electrode. This can be achieved either by introducing positive charges in the SAM or, when the SAM does not carry a charge, by labeling the enzyme with an ATTO 565 dye, which has some hydrophobic character, close to the electron entry site of the NiR.

Spectroelectrochemical investigation of intramolecular and interfacial electron-transfer rates reveals differences between nitrite reductase at rest and during turnover.

A combined fluorescence and electrochemical method is described that is used to simultaneously monitor the type-1 copper oxidation state and the nitrite turnover rate of a nitrite reductase (NiR) from Alcaligenes faecalis S-6. The catalytic activity of NiR is measured electrochemically by exploiting a direct electron transfer to fluorescently labeled enzyme molecules immobilized on modified gold electrodes, whereas the redox state of the type-1 copper site is determined from fluorescence intensity changes caused by Förster resonance energy transfer (FRET) between a fluorophore attached to NiR and its type-1 copper site. The homotrimeric structure of the enzyme is reflected in heterogeneous interfacial electron-transfer kinetics with two monomers having a 25-fold slower kinetics than the third monomer. The intramolecular electron-transfer rate between the type-1 and type-2 copper site changes at high nitrite concentration (≥520 µM), resulting in an inhibition effect at low pH and a catalytic gain in enzyme activity at high pH. We propose that the intramolecular rate is significantly reduced in turnover conditions compared to the enzyme at rest, with an exception at low pH/nitrite conditions. This effect is attributed to slower reduction rate of type-2 copper center due to a rate-limiting protonation step of residues in the enzyme's active site, gating the intramolecular electron transfer.

Probing the reactivity of different forms of azurin by flavin photoreduction.

The reactivity of a variant of the blue copper protein, azurin from Pseudomonas aeruginosa, was investigated with laser flash photolysis and compared with the reactivity of the wild-type (WT) protein. The variant was obtained by changing the Cu ligating His117 for a glycine. The mutation creates a gap in the ligand shell of the Cu that can be filled with external ligands or water molecules. The crystal structure of the H117G variant is reported. It shows that the immediate surrounding of the Cu site in the variant exhibits less rigidity than in the WT protein and that the loop containing the Cu ligands Cys112, His117 and Met121 in the WT protein has gained flexibility in the H117G variant. Flash photolysis experiments were performed with 5-deazariboflavin and 8α-imidazolyl-(N-propylyl)-amino riboflavin as electron donors to probe the reactivity of WT and H117G azurin, and of H117G azurin for which the gap in the Cu co-ordination shell was filled with imidazole. 8α-Imidazolyl-(N-propylyl)-amino riboflavin appears one to two orders less efficient as a photo-flash reductant than 5-deazariboflavin. The reactivity of the H117G variant in the absence of external ligands appears to be 2.5-fold lower than the WT reactivity (second-order rate constants of 51 ± 2 × 10(7) m(-1) ·s(-1) versus 21 ± 1 × 10(7) m(-1) ·s(-1) ), whereas the addition of imidazole restores reactivity to above the WT level (71 ± 4 × 10(7) m(-1) ·s(-1) ). The differences are discussed in terms of structural modifications and changes in reorganizational energy and electronic coupling. Database Structural data are available in the Protein Data Bank under the accession number 3N2J.

Channeling of electrons within SLAC, the small laccase from Streptomyces coelicolor.

The reduction kinetics of the fluorescently labeled small laccase (SLAC) from Streptomyces coelicolor was studied by stopped flow kinetic measurements. The tryptophan fluorescence and the emission from a covalently attached label were used to selectively follow the progress of the reduction of the trinuclear copper center (TNC) and the type-1 (T1) Cu site in the enzyme as a function of time. A numerical analysis of the kinetic traces provided new insight into the midpoint potential difference between the T1 and the TNC site as the TNC becomes stepwise charged with electrons. The change in fluorescence of the TNC as the reduction of the TNC proceeds provided evidence that the type-3 dinuclear part of the TNC becomes charged prior to the reduction of the type-2 (T2) center of the TNC. The rate of reduction of the enzyme by dithionite (DT) appeared proportional to the square root of the DT concentration with a rate constant of k(red) = 0.28 +/- 0.02 microM(-1/2) s(-1).

A FRET-based biosensor for NO detection.

In this paper we explore the use of fluorescently labeled cytochrome c peroxidase (CcP) from baker's yeast for monitoring nitric oxide (NO) down to the sub-micromolar level, by means of a FRET (Förster Resonance Energy Transfer) mechanism. The binding affinity constant (K(d)) for the NO binding to CcP was determined to be 10+/-1.5 microM. The rate of NO dissociation from the CcP (k(off)) and the second order rate constant for the NO association (k(on)) were found to be 0.22+/-0.08 min(-1) and 0.024+/-0.002 microM(-1) min(-1) respectively. The immobilization of fluorescently labeled CcP into a polymeric matrix for use in a solid state NO sensing device was also explored. The results provide proof-of-principle that labeled CcP can be successfully implemented in a fast, simple, quantitative and sensitive NO sensing device.