The primary structure of bovine adrenodoxin

Vol. 39, No. 6, 1970

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THE PRIMARYSTRUCTURE OF BOVINEADRENODCXIN Masaru Tanaka,

Mitsuru Haniu and Kerry T Yasunobu

Department of Biochemistry and Biophysics, Honolulu, Hawaii

University

of Hawaii

96822

Received May 13, 1970 SUMMARY-The amino acid sequence of bovine adrenodoxin has been determined except for the assignment of a few acid or amide groups. The protein contains 118 amino acids in the form of a single polypeptide chain. The five cysteine residues, most of which are involved in iron binding, are located at position 46, 52, 55, 95 and 98. The molecular weight of the protein from the amino acid content and the labile sulfide and the iron content is about 13,094. Although the amino acid sequences of numerous non-heme iron proteins have been determined (1), there has been no sequence determination hemeiron protein.

Erom the evolutionary

of a mammaliannon-

standpoint such a study is of importance

in order to determine whether there is homology in the sequences of the bacterial, plant and mammaliannon-heme iron proteins. established that the algal, comunon percursoral In addition,

plant and animal cy-tochromes have all

for the structure-function

The exact function protein

evolved from a

gene (2).

most essential to have the primary structure

transport

In the case of cytochrome c, it has been

and X-ray diffraction

studies, it is al-

data.

of adrenodoxin is to act as an intermediary

in a system which hydroxylates

electron-

steriods as shown as follows.

MPEFZMENTALPROCELUBE Bovine adrenodoxin-

Bovine adrenodoxin was prepared by a slight 1182

modifica-

(3)

BIOCHEMICAL

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AND BIOPHYSICAL RESEARCH COMMUNICATIONS

tion of the procedure described by Kimura (4) from bovine adrenal (kindly

glands

supplied by Dr. Yanari, Armour Pharmaceutical Co. Kankakee, Ill.).

absorbance ratio

at 276/414 mu was 0.'79 .

by the method reported by Crestfield Tryptic

and chymotryptic

doxin were hydrolyzed with either

The Cys(Cm)-adrenodoxin was prepared

et -al., --

digests-

The

(5).

About 8-10 pmoles of the Cys(Cm)-adreno-

chymotrypsin or trypsin

at 40' for 12 and

24 hours, respectively. Isolation fractionated

of tryptic

further

peptides- The enzymic digest were

on AG 5OW-X2columns using the buffer

Schroeder (6). partition

and chymotryptic

schedule reported by

When necessary, the peptides were further

chromatography or by paper electrophoresis. hydrolyzed by thermolysin and purified

the chymotryptic

and tryptic

End Group Analyses-

purified

by either

The large peptides were

by the procedures described for

pep-tides. The NH2-terminal end groups of protein and peptides

were determined by the Edmandegradative procedure (7). analyses were determined by hydrazinolysis

The carboxyl amino acid

(8) or by the use of carboxypeptidase

(9).

Sequence determinationsKoningsberger and Hill

The degradative Edmanprocedure (7) as modified by

(10) was adopted to determine the sequence of the peptide

fragments. Amino acid analyses-

The BeckmanModel 12OCautomatic amino acid analyzer

which is the commercial model of the instrument developed by Spackman, Moore and Stein (11) was used to determine the amino acid composition of the acid hydrolyzates

of the peptides.

NH, and COOH-terminal analyses of Cys(Cm)-adrenodoxin-

Three steps of the

degradative Edmanprocedure in the Cys(Cm)-adrenodoxin disclosed that the first three residues at the amino-terminal end were serine.

Hydrazinolysis

Cys(Cm)-adrenodoxin disclosed that alanine was the carboxyl-terminal

of the amino

acid (30% yield). Isolation-

The tryptic

1183

peptides which were

Vol. 39, No. 6,197O

fractionated

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on AG 5OW-X2(Biorad Labs) and eventually

form are shown in Table I.

isolated

The peptide numbers do not refer to the order

in which the peptides were eluted from the column but to their sequence starting

in a purified

from the NHS-terminal end.

location

in the

Peptide T-6-C-7 was obtained

from T-6 by the use of chymotrypsin.

TABLE I Tryptic

peptides from Cys(Cm)-adrenodoxin Sequence

Peptide no. T-l

Ser-Ser-Ser-Gln-Asp-Lys

T-2

Ile-Thr-Val-His

T-3

Phe-Ile-Asn-Arg

T-4

Asp-Gly-Glu-Thr-Leu-Thr-Thr-Lys

T-5

Gly-Lys

T-6

Ile-Gly-Asp-Ser-Leu-Leu-Asp-Val-Val-Val-Glxu-AspIle-Asp-Gly-Phe-Gly-Ala-Cys-Glu-Gly-Thr-Leu-Ala-Cys-Ser-TkrCys-His-lku-Ile-Re-Glu-Gln-His-Ile-Phe-Glu-Lys

T-6-C-1

Ala-Cys-Ser-Thr-Cys-His-Leu-Ile-Phe

T-7

Leu-Glu-Ala-Ile-Thr-Asn-Glu-Glu-Asn-Asn-Met-Asx-Glx-Leu-LeuAsp-Leu-Ala-Tyr-Gly-Ieu-Thr-Asp-Arg

T-8

Ser-Arg

T-9

Ieu-Gly-Cys-Gin-Ile-Cys-Ieu-Thr-Lys

T-10

Ala-Met-Asp-Asn-Met-Asp-Thr-V&l-Arg

T-11

Val-Pro-Asp-Ala-Val-SeryAsp-Ala

Chymotryptic

pep-bides of Cys(Cm)-adrenodoxin- The various chymotryptic

peptides which were obtained from Cys(Cm)-adrenodoxin by chromatography on AG 5OW-X2are listed

in Table II.

The various peptides designated by the symbol

Th, were obtained by the use of thermolysin 1184

(l-2).

BIOCHEMICAL

Vol. 39, No. 6, 1970

AND BlOPHYSlCAL

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TABL3 II Chymotryptic peptides from Cys(Cm)-adrenodoxin . Peptide no.

Sequence

C-l

Ser-Ser-Ser-Gln-Asp-Lys-Ile-Thr-Val-His

c-2

Ser-Ser-Ser-Gin-Asp-Lys-Ile-Thr-Val-His-Phe

c-3

Ile-Asn-Arg-Asp-Gly-Glu-Thr-Leu

c-4

Thr-Thr-Lys-Gly-Lys-Ile-Gly-Asp-Ser-Leu-Leu-Asp-Val-Val-ValGlx-Asx-Asn-Leu-Asp-Ile-Asp-Gly-Phe-Gly-Ala-Cys-Glu-Gly-ThrLfXl

C-b-Th-1

Thr-Thr-Lys-Gly-Lys

C-4-Th-2

Ile-Gly-Asp-Ser-Leu

C-k-Th-3

ku-Asp-Val-Val-Val-Glx-Asx-Asn

C-4-Th-4

Leu-Asp-Ile-Asp-Gly

C-4-Th-5

Phe-Gly-Ala-Cys-Glu-Gly-Thr-Leu

C-4-Th-6

Leu-Asp-Ile-Asp-Gl~~-Phe-Gly-Ala-Cys-Glu-Gly-Thr-Leu

C-5

Ala-Cys-Ser-Thr-Cys-His

c-6

Leu-Ile-Phe

c-7

Glu-Gln-His-Ile-Phe

c-8

Glu-Lys-Leu-Glu-Ala-Ile-Thr-Asn-Glu-Glu-Asn-Asn-Met-Asx-GlxLeu-Leu-Asp-Leu-Ala-Tyr

C-8-Th-1

Glu-Lys-Ieu-Glu-Ala

C-8-Th-2

Ile-Thr-Asn-Glu-Glu-Asn-Asn-Met-Asx-Glx-Leu

C-8-Th-3

Leu-Asp

C-8-Th-4

Leu-Ala-Tyr

c-9

Glu-Gln-His-Ile-Phe-Glu-Lys-Leu

c-10

Gly-Leu-Thr-Asp-Arg-Ser-Arg-Ieu

C-11

Thr-Asp-Arg-Ser-Arg-Leu

c-12

Gly-Cys-Gln-Ile-Cys-Leu

c-13

Thr-Lys-Ala-Met-Asp-Asn-Met-Asp

c-14

Thr-Val-Arg-Val-Pro-Asp-Ala-Val-Ser-Asp-Ala 1185

Vol. 39, No. 6,197O

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AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Complete amino acid sequence- The NH2-terminal peptide from the chymotryptic

and tryptic

digests were readily

identifiea

due to the Ser-Ser-Ser-

sequence. Also, the COOH-terminal peptide from the two digests were easily spotted since these peptides were the only ones which contained a COOH-terminal alanine residue.

There are overlaps between the try-ptic

and chymotryptic

which allowed the uneqivocal placement of the remainder of the peptides.

peptides The

amino acid sequence of bovine adrenodoxin established from the present study

Amino acid seguence of --- bovine adrenodoxin

Fig. I

Ser-Ser-Ser-Gln-Asp-l;ys-Ile-Thr-Val-His-Phe-Ile-ASn-Arg-Asp-Gly-Glu-Thr-Leu-T~-T~20 &--

T-l -YT-2LT-3

p-

-

c-2 -

T-4 C-3 -

L~s-Gly-Lys-Ile-Gly-Asp-Ser-Leu-Leu-Asp-Val-Val-Val-Glx-Asx-Asn-Leu-Asp-I~~-Asp-Gly 30 -T-5+-

T-6

-C-4-Th-l--S)C-C-4-Th-2

-

LA

' C-4-Th-3 ,-+

C-4-Th-4---r(

Phe-Gly-Ala-Cys-Glu-Gly-Thr-Leu-Ala-Cys-Ser-Thr-Cys-His-~u-Ile-Phe-Glu-Gln-His-Ile 60

50 c-4 T

b--

c-5---4+-c-6

-+-c-7

-

C-4-Th-5--3

1Jhe-Glu-Lys-Leu-Glu-Ala-Ile-Thr-Asn-Glu-Glu-Asn-Asn-Met-Asx-GLx-l;eu-Leu-Asp-Leu-Ala80 70 T-7 c-8

C-8Th-2.-L'C-8-Th-3+-8-Th.

&-C-8-Th-1.e

Tyr-Gly-Leu-Tkr-Asp-Arg-Ser-Arg-Leu-Gly-Cys-G~-Ile-Cys-Leu-Thr-Lys-Ala-Met-Asp-Asn100 T-8+T-g", +-

c-10-*-

c-12 .--+-

4* Met-Asp-Thr-Val-Arg-Val-Pro-Asg-Ala-Val-Ser-Asp-Ala 110

1186

C-13-

T-10

Vol. 39, No. 6, 1970

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AND BIOPHYSICAL RESEARCH COMMUNICATIONS

is shown in Fig. 1.

However, it

should be noted that residues 35. 36, 78

and 79 may be either

in the form of the free acids or amides. DISCUSSION

The sequence studies of bovine adrenodoxin indicate weight of the protein

is 13,094.

that the molecular

This takes into account the 118 amino acid

residues. two moles of iron and labile

sulfide.

This differs

somewhatfrom the

published molecular weight of 12,000 and 97 amino acid residues as reported by Kimura (3). The primary structure bacterial

of bovine adrenodoxin showedno homology with the

and plant non-heme iron proteins nor with mamalian hemeproteins.

Thus, the mammaliannon-heme iron proteins have evolved from a separate gene which has no counterpart

or has undergone numerousmutations so that the homology

is no longer evident in the bacteria evidence for gene duplication

and plants.

In addition there is no

which has been observed amongthe bacterial

non-heme iron proteins. As far as the primary structure

of adrenodoxin is concerned, there are a

number of unusual features such as the presence of repeating amino acid residues for example, Ser-Ser-Ser and Val-Val-Val.

The presence of the Cys-x-x-Cys

which is present in the many of the non-heme iron

proteins sequenced thus far,

suggests that residues 52, 54, 95 and 97 may be involved details

of the present s.tudy will

in iron binding.

The

be published elsewhere when the primary structure

has been completed.

Acknowledgements- This work was supported by grants from the National Institutes of Health, GM 16228-02

and GM 16784-01 REFERENCES

1.

Dayhoff, M. O., Atlas of protein ---

sequence -and structure,

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Margoliash, E., and Fitch,

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Kimura, T.,

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BIOCHEMICAL

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