Development of a fast scanning IR spectrophotmeter and its application to oxygenase systems.

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Aerobic organisms utilize molecular oxygen ( <O_2> ) in many ways. Such oxygen utilizing reactions include various oxidase and oxygenase reactions, and superoxide-producing reactions. Under physiologic conditions, however, molecular oxygen is mainly in an inactive triplet groud state which usaully requires an "activation" to react with singlet organic molecules. Thus, the mechanisms for activation of molecular oxygen has been one of the main themr in biochrmistry. In the present study, we developed a new type of IR spectrophotometer which enables us to measure O-O stretching frequency around 1100 <cm^(-1)> of molecular oxygen bound to iron in heme-containing oxygenases and oxidases. Since such oxygen complexes are all unstable reaction intermediates, it requires fast scanning together with high sensitivity, which are not fullfilled by any dispersive and/or FT type of IR spectrophotometer. For this purpose, we succesfully developed a new type of machine by employing MCT, high frequency chopper and high speed scanning device with special soft wear systems. The spectrophotmeter system made us possible to measure the O-O stretching of oxygen in oxy-CoMb (4 mM) within 7 minutes at 10゜C. In preliminary experiments, we also succeeded in measuring the O-O stretching of oxy-P450cam which was stabilized by utilizing the diacetylheme-substituted enzyme.