Objective
Archaea, bacteria and the mitochondria of eukaryotes contain the aa3-type terminal oxidase, responsible for the reduction of molecular oxygen to water and hence for much of biological energy metabolism. This oxidase, together with 2 alternative types of terminal oxidase found in some organisms, belong to a superfamily of terminal oxidases. Many species of bacteria contain the enzyme nitric oxide reductase (NOR), which reduces nitric oxide (NO) to nitrous oxide. One of the recent surprising findings has been that NOR belongs to the superfamily of terminal oxidases. By implication, their common ancestor of more than 2 billion years ago may have been a protein resembling NOR. Evolutionary divergence subsequent to the appearance of oxygen in the atmosphere may have led to the branch of terminal oxidases.
Much of the structure and function of the terminal oxidases is known, but the catalytic mechanism of oxygen reduction and its coupling to proton pumping are not yet settled. This is partly because of a lack of functional enzymes with differences in the parameters that are critical for these properties.
NOR may be such an enzyme: against the backdrop of the similarities in structure and function, NOR differs in two functional and two structural aspects from the oxidases. The functional differences are that NOR reduces NO at a higher rate than oxygen (whereas the oxidases reduce NO at a lower rate than oxygen) and that it appears to pump fewer if any protons. The structural differences are the presence of iron rather than copper in the binuclear metal center that is responsible for the reduction of oxygen or NO and the precise amino acid residues that reside in the alpha helices around the catalytic center.
It is the objective of this proposal to determine whether these structural differences are responsible for the functional differences and hence generate a deeper insight into the structural evolution and catalytic mechanism of the superfamily of terminal oxidases.
We shall aim to achieve this objective by determining the structure of NOR by X-ray crystallography and by unraveling the molecular mechanism of NO reduction by a combination of advanced spectroscopic and biochemical techniques. All of this will be done in continuous comparison with the already known properties of the oxidases.
The strategy is that from the differences between NOR and the oxidases much more of the mechanism of either will be learnt. The comparative analysis will culminate in the construction of enzymes that are hybrids between NOR and oxidases: (i) by changing key amino acids by site directed mutagenesis, (ii) by substituting copper for iron in the dinuclear center of NOR, and (iii) by changing NOR into an oxidase by guided evolution. By analyzing at which extent of modification NOR begins to reduce oxygen more rapidly than NO, and begins to pump protons efficiently, we shall greatly expand our understanding of the molecular mechanism of this most important class of enzymes.
Fields of science (EuroSciVoc)
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
- natural sciences biological sciences microbiology bacteriology
- natural sciences earth and related environmental sciences geology mineralogy crystallography
- natural sciences chemical sciences organic chemistry amines
- natural sciences biological sciences biochemistry biomolecules proteins enzymes
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Coordinator
1081 HV Amsterdam
Netherlands
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