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... Show moreMonitoring 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 E0. 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. Show less
Pradhan, B.; Engelhard, C.; Mulken, S. van; Miao, X.; Canters, G.W.; Orrit, M. 2019
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... Show moreFluorescent 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. Forster 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 angstrom, but not when it is at 29.1 angstrom from the Cu. For Cu-II, PET from label to Cu occurs at a rate of (4.8 +/- 0.3) x 10(4) s(-1) and back at (0.7 +/- 0.1) x 10(3) s(-1). With Cu-I the numbers are (3.3 +/- 0.7) x 10(6) s(-1) and (1.0 +/- 0.1) x 10(4) 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. Show less
Mucibabic, M.; Apetri, M.M.; Canters, G.W.; Aartsma, T.J. 2016
Rede uitgesproken door Prof. Dr. G.W. Canters ter gelegenheid van zijn afscheid als hoogleraar in de Biofysische Chemie aan de Universiteit Leiden op vrijdag 16 oktober 2015
The recent exciting developments concerning the structure and function of the Cu-A site in cytochrome c oxidase (COX) and nitrous oxide reductase are reviewed. The XRD structures of a mitochondrial... Show moreThe recent exciting developments concerning the structure and function of the Cu-A site in cytochrome c oxidase (COX) and nitrous oxide reductase are reviewed. The XRD structures of a mitochondrial and a bacterial COX have been reported in 1995, In COX, the Cu-A centre is located in subunit II and is involved in electron transfer from a cytochrome c donor to the oxygen binding centre of the protein. The structural studies on the enzyme have revealed the coordination geometries of the many metal centres of COX, including the Cu-A site. A combination of spectroscopic and mutagenesis studies on the Cu-A centre led to the correct prediction of the structure of this unique copper centre. The construction of a Cu-A site in type 1 blue copper proteins is briefly discussed. Show less
A small library of truncated neomycin-conjugates is prepared by consecutive removal of 2,6-diaminoglucose rings, oxidation-reductive amination of ribose, oxidation-conjugation of aminopyridine... Show moreA small library of truncated neomycin-conjugates is prepared by consecutive removal of 2,6-diaminoglucose rings, oxidation-reductive amination of ribose, oxidation-conjugation of aminopyridine/aminoquinoline and finally dimerization. The dimeric conjugates were evaluated for antibacterial activity with a unique hemocyanin-based biosensor. Based on the outcome of these results, a second-generation set of monomeric conjugates was prepared and found to display significant antibacterial activity, in particular with respect to kanamycin-resistant E. coli. Show less
