CYP199A4, is a bacterial P450 enzyme involved in the biodegradation of many organic compounds under aerobic and anaerobic conditions. This enzyme utilizes a class I electron transfer system which requires two electrons delivered via the electron donor (NADH) to the P450 enzyme via two redox partner proteins, ferredoxin reductase (HaPuR) and ferredoxin (HaPux)^[1]. The aim of our study is to understand the interactions between these electron transfer proteins with the P450 enzyme in order to reveal the structural and functional basis of catalysis. Conformational changes in these proteins as they interact can be monitored using Raman Spectroscopy and Quartz Crystal Microbalance (QCM). Raman spectroscopy can provide information of the local chemical environment (i.e. oxidation and spin state) based on the changes of molecular vibration modes^[2]. In addition, QCM measures mass uptake/loss as the protein adsorbed on the sensor and can define the resulting organisational properties of the proteins as they interact ^[3].

CYP199A4 shows no change in the present and absent of substrate using Raman spectroscopy and QCM. This suggests that CYP199A4-substrate interaction does not alter the structure of the active site or the global protein conformation. We have also monitored the interaction of CYP199A4 with the two redox partner proteins (HaPux and HaPuR) using QCM under both reduced and oxidized conditions. An increase in HaPuR adsorption to the CYP199A4-HaPux complex previously deposited on the QCM sensor was observed. However, if the three proteins are mixed, presumably forming a CYP199A4-HaPux-HaPuR complex in solution, this effect has diminished. Hence, this protein-protein interaction could be a redox dependent regulatory mechanism. We will present our data illustrating the combined use of QCM and Raman spectroscopy to provide some new insights into the regulation of bacterial enzymes function through protein-protein interactions.