Skip to main content
ARS Home » Research » Publications at this Location » Publication #106326
Title: CYTOCHROME CM IN SYNOCHOCYSTS 6803 DETECTION IN CELLS, OVEREXPRESSION IN E. COLI, AND PHYSICAL CHARCTERIZATION

Author
item CHO, Y - PLANT BIOLOGY UOFI URBANA
item PAKRASI, H - WASHINGTON UNIV STL MO
item WHITMARSH, CLIFFORD

Submitted to: European Journal of Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/14/2000
Publication Date: N/A
Citation: N/A

Interpretive Summary: Photosynthesis provides the energy and building blocks necessary for plant growth. The photosynthetic process depends on a series of electron transport reactions that convert light energy into chemical energy, primarily in the form of carbohydrates. To improve photosynthetic performance, we need to understand the function and structure of the electron transport proteins. This knowledge opens the way to genetically manipulate photosynthesis for optimization under diverse environmental conditions. With this aim we have introduced the gene for a metal containing protein from a photosynthetic organism into the bacterium E. coli. We showed that the gene was expressed, producing a heme-containing protein that had never been seen before. We have characterized the physical properties of the protein and are working to determine its three-dimensional structure. This information will allow us to discover its role in photosynthesis, with the goal of improving photosynthetic performance.

Technical Abstract: Based on DNA sequence data and measured mRNA levels a novel c-type cytochrome, cytochrome cM, has been predicted to exist in the cyanobacterium Synechocystis 6803. The precursor protein consists of 105 amino acids with a characteristic heme-binding motif and a hydrophobic tail located at the N-terminal end that is proposed to act as either a transit sequence or a membrane anchor. For the first time we report the detection of cytochrome cM in Synechocystis 6803 using Western analysis. The soluble portion cytochrome cM has been over expressed in E. coli in two forms one with a poly-histidine tag to facilitate purification and one without the histidine attachment. The over expressed protein binds heme, exhibiting an absorption peak in the Soret-band at 415 nm and a peak in the alpha-band at 550 nm. The reduced minus oxidized extinction coefficient of cytochrome cM is 23/(mM cm) for the a-band peak (550 - 535 nm). The isoelectric point of cytochrome cM is 5.6 (without the histidine tag), which is significantly lower than the pI of 7.3 predicted from the gene sequence. The oxidation/reduction midpoint potential of cytochrome cM is 150 mV at pH 7.1. This work opens the way for determining the three-dimensional structure of cytochrome cM and for establishing its function in cyanobacteria.