ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 1, April, pp. ZlS-226,198sThe Amino Acid Sequence of Cytochrome CATHLEENc 553from MicrocystisL. COHN, MARK A. HERMODSON,AND DAVIDaeruginosaW. KROGMANN’Department of Biochemistry, Purdue University, West Lqfavette, Indiana 47907 Received August 22,1988, and in revised form November 14,1988Cytochrome cs3 is an electron donor to P700 in the photosynthetic electron transfer chain of cyanobacteria and eukaryotic algae. We have purified this cytochrome from the cyanobacterium Microcystis aerugirwsa and determined its amino acid sequence.
When the amino acid sequence of this protein is compared to sequences of cytochromes c5% from other organisms, one sees that the evolution of net charge is more pronounced than the evolution of overall structure, further documenting a pronounced shift in the isoelectric point of this protein during the evolution of cyanobacteria. Cyanobacteria and algae also contain cytochrome cSo (M, 15,500) which is quite different from cytochrome c553(M, 10,500). When the amino acid sequence of cytochrome c553is compared to that of cytochrome c550,two regions of similar sequence are recognized.
Each letter stands for a different amino acid. Each column is a location on the protein chain. Note: Locations where the amino acids are identical in all eight mammals are not shown. Use the row labeled Human as your control. Compare the sequence for the bear to the sequence for humans. When you find a difference in the bear sequence, highlight it. What are amino acids, and what is an amino acid sequence? This lesson answers those questions and explains the structure, importance, and role of amino acids in protein synthesis.
O 1989 Academic Press, Inc.Cytochrome cm3 transfers electrons from cytochrome f to P700, replacing the copper protein plastocyanin which catalyzes this reaction in higher plant chloroplasts. This replacement occurs in cyanobacteria and in some eukaryotic algae that are experiencing copper deprivation (1). Natural waters often become deficient in copper and these oxygenic photosynthetic organisms sustain their activities by substituting the cytochrome for the copper containing plastocyanin. Earlier work from this laboratory has shown that these two functionally interchangeable proteins show parallel variations in their isoelectric points depending on the genus from which the proteins have been isolated (2). The isoelectric points of cytochrome c553 and plastocyanin in eukaryotic algae and the simpler forms of cyanobacteria are, like the plastocyanins of higher plants, acidic (p13.8 to 6.5) while those of the more complex cyanobacteria are basic (PI 7.5 to 9.5).
It is of interest to compare the structures of the acidic and basic cytochromes in order to see what regions are responsi1 To whom correspondence should be addressed. 219ble for the change in isoelectric point and to recognize features important to function and to evolutionary history. An important shift from basic to acidic residues in the region between residues 67 and 77 (as in Fig. 2) in cytochrome c553was noted earlier (2) and is confirmed in the sequence reported here. Cytochrome c5%is a member of the Class I c type cytochromes (3). These cytochromes have low-spin iron, His + Met heme coordination, heme near the N-terminus, and lengths of 80 to 120 amino acids.
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Mitochondrial respiratory cytochrome c is the prominent member of this class with many other members found in bacteria. All are related by similarity in amino acid sequence. The cytochrome c5% described in this paper is seen to contain regions of sequence that show striking similarity to regions of a cytochrome c550 from the same source. This latter cytochrome, whose sequence is described in an accompanying paper (4), is different from all of the Class I c cytochromes in size, redox potential, and heme binding. EXPERIMENTAL PROCEDURES Materials. Micmcgstis aeruginosa was collected from the Potomac River at Pohick Bay in 1983 as has 0003-9861/89 $3.00 Copyright All rights0 1989 by Academic Press.
Of reproduction in any form reserved.220COHN,HERMODSON,ANDKROGMANN10 20 Asp Gly Ala Ser Ile Phe Ser Ala Asn Cys Ala Ser Cys His Met Gly Gly Lys Asn Val N terminus CB-1 GB-2-I I-30 40 Val Asn Ala Ala Lys Thr Leu Lys Lys Glu Asp Leu Val Lys Tyr Gly Lys Asp Ser Val CB-250Glu Ala Ile I-60Val Thr Gln Val Thr Lys Gly Met Gly Ala Met Pro Ala Phe Gly Gly Arg CB-2 +-CB-3 1 V8-2JOLeu Ser Ala Glu Asp Ile -V8-2,-i80Glu Ala Val Ala Asn Tyr Val Leu Ala Gln Ala Glu Lys Gly Trp CB-3FIG. The amino acid sequence of cytochrome c 653. The fragments labeled CB-1, CB-2, and CB-3 were obtained by cyanogen bromide cleavage and the fragment VS-2 was isolated from a digest done with Staphylococcus aurem VS protease.been described (5). The cells were broken by freezing and thawing three times. The broken cells were easily separated from the soluble extract by filtration through cloth. The cell mass was extracted repeatedly to remove nearly all of the blue pigment which served as a convenient indicator for removal of the soluble proteins (6).
The aqueous extract was filtered through paper (Schleicher & Schull, No. 595) to remove small, green membrane fragments and centrifuged at 27,OOOg in a continuous flow Sorvall centrifuge at a flow rate of 200 ml/min to remove phycobilisome fragments. The soluble proteins were pumped through a Pellicon Millipore No.
PTHK00005 filter (M, 100,000 cutoff) which removed 95% of the phycobiliprotein. Next the extract was concentrated on a Millipore PTGA00005 filter (I& 10,000 cutoff) to reduce the volume.
The concentrated extract was brought to 50% saturation with (NH&SO, and centrifuged at 10,OOOg for 10 min to remove the lasttracesofphycocyanin.The dialyzed against fuged as above,supernatantsolutionwas5 mM Tris buffer, pH 7.8, and centrithen loaded on a DE52 column (35X 250 mm) equilibrated with 5 mM Tris buffer, pH 7.8. The individual proteins were eluted as previously described (7).
The cytochrome cm fraction was loaded on a DEAE Sephadex A-25 column (25 X 150 mm) equilibrated with 5 mM Tris buffer, pH 9, and was eluted with 0.05 M NaCl. The protein was then passed through a Sephadex G-50 gel filtration column equilibrated with 50 mM ammonium bicarbonate. A final purification was achieved by HPLC, using a Synchropak RP-R C-18 reverse phase column (250 X 1.4 mm) eluted with a 0 to 60% acetonitrile gradient in 0.1% trifluoroacetic acid.
The cytochrome eluted as a symmetrical peak at 42% acetonitrile. The same column was used for peptide separations.
Elution was monitored at 230 nm at a flow rate of 1 ml/min. Solvents for HPLC were from Burdick & Jackson. Cyanogen bromide was from Pierce; all other chemicals were reagent grade. Staphylococcus aureus V8 protease was obtained from Pierce and endoproteinase Lys-C from Boehringer. Enzymatic cleavages.