High-field EPR studies on reaction intermediates of NO Synthase

• Applicant: Dr. Friedhelm Lendzian
• Subject Area: Biophysics
• Term: from 2002 to 2005
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 5381321

The key intermediate in the reaction cycle of the heme enzyme nitric oxide synthase (NOS) which catalyses the synthesis of the signal molecule NO in biological systems is generally believed to be a Fe(IV)=O porphyrin-p-cation radical. This so called Compound I species in the NOS reaction cycle has been proposed from analogy with other enzymes but not prepared and spectroscopically characterized so far. Our first X-band EPR studies on rapid-freeze quenched iNOS samples which have been prepared by reaction with peroxy acetic acid (peroxide shunt) for 8 ms indicate the presence of a protein side chain radical as well as a ferryl (Fe(IV)) porphyrin-p-cation radical. We propose to investigate the nature of the protein radical by multi-frequency EPR (in particular by W-band EPR) of NOS intermediates prepared by the rapid-mixing/freeze-quench technique for reaction times ranging from the millisecond up to the minute time scale. We will improve the preparation procedure of small freeze-quenched W-band (NOS) samples, a technique which has been recently developed in the laboratory of the Institute of Physics, Medical University of Lübeck. This technique will then be also available for other projects in this priority programme, where short-lived intermediates must be trapped.

• DFG Programme: Priority Programmes
• Subproject of: SPP 1051: Hochfeld-EPR in Biologie, Chemie und Physik