Probing Local Solvation Environments in H-NOX Proteins using 4-Cyano-L-Phenylalanine

Abstract

The production of reactive oxygen species (ROS) through external factors can lead to various microbial processes. As a physical means, cold plasma has a wide range of applications in the areas of bio-engineering and bio-medicine. It can produce ROS and affect microorganisms effectively, resulting in the effects of stimulation, inactivation, apoptosis and necrosis of cells. Cytochrome c is a common globular protein located in the inner mitochondrial membrane of cells, and its function is to transfer electrons in the mitochondrial respiratory chain between cytochrome c reductase (complex III) and cytochrome c oxidase (complex IV). Under oxidative stress, cytochrome c can be released from the inner mitochondrial membrane into the cytoplasm so that it is also regarded as a pro-apoptotic factor. In this work we employed atmospheric roomtemperature plasma (ARTP) to treat yeast, and employed Raman microspectroscopy to monitor the state changes of yeast cells under plasma irradiation. As a result, we found that the Raman signals attributable to the reduced yeast cytochrome c at 750 cm À1 , 1128 cm À1 , 1310 cm À1 , 1585 cm À1 decreased with the increase of discharge time, and at a certain time after the plasma irradiation, the Raman peak at 1636 cm À1 which is attributed to oxidation state of cytochrome c became prominent and enhanced. We also examined the apoptosis of plasma irradiated yeast cells by Annexin V-FITC/PI, and found that the number apoptotic cells increased gradually with the increase of plasma irradiation time. This work thus demonstrates that we can utilize Raman microspectroscopy to track the vital redox processes of some important biomolecules such as cytochrome c in single yeast cells, which thus may help us to understand the cold plasma irradiation induced biological effects in the microbes.