Single molecule studies of redox enzymes

This presentation will deal with copper proteins. The body of an adult human male contains about 80 mg of Cu and this amount derives from about 30 different copper proteins. In the absence of copper most life forms including man, would be non-existent. Cu proteins almost exclusively are involved in electron transfer processes or the catalysis of redox processes most of them involving oxygen. They occur outside the cellular matrix or within vacuoles and organelles inside it. Depending on their structural characteristics six to seven distinct types of copper site have been distinguished, each connected with its own particular type of chemistry or chemical reactivity. The most common types are the so-called type-1 and Type-2 sites, the former being operational in electron transfer the latter in enzymatic redox chemistry. Azurin will be presented and discussed as the proto-type-1 Cu containing protein and nitrite reductase (NiR) as the primary example of an oxido-reductase containing a type-1 as well as a type-2 site. Attempts to engineer azurin constructs by which the process of electron transfer can be followed in minute detail, will be presented. A variety of spectroscopic techniques ranging from magnetic resonance to scanning probe microscopy and single molecule spectroscopy will be reviewed. The case of NiR will be used to illustrate how single molecule techniques can be employed to characterize individual enzyme molecules and discern individual differences in their mechanistic behavior.


 About Prof. Gerard W. Canters :

Professor Gerard W. Canters received his primary degree in Chemistry/Chemical Physics from the University of Amsterdam. He obtained his PhD from the University of Nijmegen under the direction of Professor E. de Boer. In 1969 he received the Royal Dutch Shell award for young chemists. After a post-doctoral fellowship at the University of North Carolina, Chapel Hill he joined the academic staff of the Physics Department at Leiden University in 1972. In 1976 he moved to the Organic and in 1980 to the Inorganic Chemistry Department at Leiden. In 1982 he established the Metallo-protein Group and in 1992 he was appointed Professor of Biophysical Chemistry in the Biochemistry Department at Leiden University. His research interests have predominantly focused on the molecular characterization of redox enzymes and proteins involved in biological electron transfer. He has used nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopies, X-ray crystallography, electrochemistry, and protein engineering to study type 1 copper and multinuclear copper proteins. 1987 was a pivotal year for copper protein research when he cloned the gene for the protein Azurin. This opened up vast possibilities for the study of structure / function relationships through site-directed mutagenesis. In particular, it became possible to determine how the features of the metal-binding site contribute to the redox state and the electron transfer capabilities of the protein. His current research interests are concerned with the study of biological redox processes by Förster-resonance-energy transfer (FRET) techniques. Recently, he has shown that FRET can be used to detect the redox state of a protein and to study the kinetics of single enzyme molecules. He has fulfilled various guest professorships, among them visiting professorships at the Royal Danish Veterinary University and the University of Salerno, a visiting scholarship at the Tata Institute for Fundamental Research, Mumbai, and a 'Kugie and Bert Vallee' professorship at Oxford University. Since 2005 he is connected with the Leiden Institute of Physics at Leiden University.