Purification And Functional Studies Of The Electron Carrier Cytochrome Cx From Roseiflexus Castenholzii

Cytochrome c(cyt c)is a kind of redox protein widely existing in organisms.It participates in the electron transport chain of photosynthesis and respiration,and forms an electron transport network with many complexes.In the photosynthetic chain of many photosynthetic bacteria,cytochrome c is usually responsible for electron transfer on the periplasmic side of the membrane.In this study,Roseiflexus castenholzii,one of thermophilic photosynthetic bacteria,was taken as the research object.It was found in previous studies that the photosynthetic electron transport chain of Roseiflexus castenholzii lacked the commonly existed soluble C-type cytochrome to complete the circulating electron transfer pathway in the periplasmic side of the membrane.In this study,a reduced cytochrome c named RfxCx was directly extracted and purified from Roseiflexus castenholzii.It is the first soluble electron carrier cytochrome c found in Roseiflexus castenholzii.The redox activity of RFXCX was determined by spectrophotometry and enzyme activity assay.It was shown that RfxCx can accept electrons from the oxidation of methyl naphthoquinone-4(MK-4)by the substitution complex ACIII,indicating that RfxCx is a novel electron acceper for Rfx ACIII.At the same time,we obtained active cytochrome Cx by heterologous expression in E.coli.Circular dichroism analysis showed that there was no significant difference in secondary structure content between heterologous CX and naturally extracted CX.Based on the above results,we further carried out crystal growth screening experiments on heterogenously expressed Cx in order to obtain a single crystal that could analyze its three-dimensional spatial structure and lay a foundation for further research on its structure and the mechanism of action of electron transport.