The soluble low potential cytochrome c from the unicellular cyanobacterium Synechocystis PCC 6803

Beng-Hoon Charmaine Kang Grant, Purdue University

Abstract

A novel purification strategy was developed for the isolation of homogeneous soluble low potential cytochrome c from the unicellular cyanobacterium Synechocystis PCC 6803. The purification procedure involves uncomplicated manipulations. The low potential cytochrome c was separated from the major group of contaminants, the phycobiliproteins, by first subjecting the initial crude protein extract to a two-step $\rm(NH\sb4)\sb2SO\sb4$ precipitation followed by filtration of this extract through a Millipore Pellicon filtration unit. The Millipore Pellicon retained almost all the phycobiliproteins aggregates. The Millipore Pellicon filtrate which contained the low potential cytochrome c was visibly devoid of any blue pigment. This is the key to obtaining homogeneous low potential cytochrome c from Synechocystis PCC 6803. The low potential cytochrome c was further purified by chromatography on a DEAE-52 column and finally, by SDS-PAGE. The molecular weight of the cytochrome was determined by SDS-PAGE to be approximately 16 kDa. The cytochrome has a pI value of 4.5 as determined by native IEF. Out of the 21 N-terminal amino acids determined for this cytochrome, 7 were conserved in the low potential cytochromes c from three other cyanobacteria. The gene for the low potential cytochrome c (petK) was isolated by screening a genomic library of Synechocystis PCC 6803. The probe used for the screening was designed based on residues 11 through 16 from the N-terminal amino acid sequence and based on codon usage observed for many genes of Synechocystis PCC 6803. The petK gene begins with a GTG and encodes for a precursor form of the cytochrome (160 amino acids) from which a mature protein (135 amino acids) is generated by proteolytic cleavage. The leader peptide resembles typical prokaryotic signal sequences. The nucleotide sequence of the mature petK protein contains two regions with sequence similarity to two regions in the gene for the high potential cytochrome c$\sb6$. The sixth iron ligand can be identified with a conserved histidine. The low potential cytochrome c could be reduced by ferredoxin II. The heme of the cytochrome is flanked by lysines which may be involved in orienting the ferredoxin near the site of electron transfer. The petK gene is expressed as a monocistronic message. There are two apparent transcription start sites which may suggest the transcription of two slightly different transcripts from the same gene. Cells of Synechocystis PCC 6803 that were deprived of oxygen and light for 18 hours have higher level of petK transcripts. This indicates that the expression of the petK gene is probably regulated at the transcription level.

Degree

Ph.D.

Advisors

Krogmann, Purdue University.

Subject Area

Biochemistry

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