BIOCHEMICAL

Vol. 76, No. 4, 1977

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

AMINO ACID SEQUENCE OF THE HEME 5 SUBUNIT OF BOVINE HEART CYTOCHROME OXIDASE AND SEQUENCE HOMOLOGY WITH HEMOGLOBIN Masaru Department

Tanaka,

Mitsura

Haniu,

of Biochemistry-Biophysics,

and Kerry

University

Honolulu,

Hawaii

T. Yasunobu

of Hawaii

School

of Medicine

96822

and C. A. Yu, L. Yu, Y. H. Wei, Laboratory State

Received

of Bioenergetics

University

April

and Tsoo E. King

and Department

of New York

at Albany,

of Chemistry

Albany,

New York

12222

17,1977

SUMMARY: The amino acid sequence of the 11.6 K dalton heme 2 subunit of bovine heart cytochrome oxidase has been completed and is presented here. The sequence investigation has established the positions in the protein of all the possible heme ligands, namely cysteine, methionine, histidine and lysine residues. However, the isolation conditions may have caused the heme 2 to migrate from its original site or the heme is caged by peptides as pointed out in Reference 6. The sequence of the heme a subunit and the S-chain of hemoglobin shows homology. It is possible that these two proteins have arisen from a common ancestor in the distant past. There

are

several

cytochrome

oxidase

(l-4)

laboratories

have

jointly

amino

acid

sequence

laboratories but

investigation,

the complete

which

heme a is

contains

and evolutionary

in

few on the amino

engaged

of the

involved

in

amino

acid

reported.

relationship

of this

acid

the latter

"copper-subunit"

the studies sequence

and have

Certain protein

(5).

of the "11.6

aspects with

of the other

of Our

investigation.

reported

of the oxidase sequence

of subunits

the partial In the present

K dalton"

subunit

structure-function

hemoproteins

are dis-

cussed. MATERIALS -~ AND METHODS Bovine cytochrome reported previously (6). described by Crestfield methylated subunit was to Klotz (8). Tryptic derivative was carried tographic methods. The position (9), sequence

Copyright All rights

oxidase and the 11.6 K heme subunit were isolated as The isolated heme 5 subunit was carboxymethylated as et al. (7). The succinyl derivative of the carboxyprepared by reaction with succinic anhydride according digestion as well as CNBr cleavage of the protein out and the peptides were purified by paper chromaprocedures for the determination of the amino acid comdetermination (lo), NH2 and COOH-terminal amino acid

0 1977 by Academic Press, Inc. of reproduction in any form reserved.

1014 lSSN 0006-291X

Vol. 76, No. 4, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1 5 10 Ser-His-Gly-Ser-His-Glu-Thr-Asp-Glu-Glu-Phe-~p-~a-Arg-Trp-Val-~r-Tyr-Phe-Asn-

15

20

(Sequencer

I I

I I

ST1

ST2

25 30 Lys-Pro-Asp-Ile-Asp-Ala-Trp-Glu-Leu-Arg-Lys-Gly-Met-Asn-Thr-Leu-Val-Gly-Tyr-Asp-

35

40

Sequencer CNBr 1

CNBr 2

I

T-2

IT-3l I I I

ST2

T-4 ST3

45 50 Leu-Val-Pro-Glu-Pro-Lys-Ile-Ile-Asp-Ala-Ala-Leu-Arg-Ala-Cys-Arg-Arg-Leu-Asn-Asp-

55

60

Sequencer CNBr 2 T-4

T-5

I

ST3

I

T-6

I I

ST4

65 70 Phe-Ala-Ser-Ala-Val-Arg-Ile-Leu-Glu-Val-Val-Lys-Asp-Lys-Ala-Gly-Pro-His-Lys-Glu-

75

,T-7, I I lST51 I I

T-8 ST6 80

CNBr 2 T-8

I

ST6

I

T-9

JT-10 I ST7

, I

85 90 Ile-Tyr-Pro-Tyr-Val-Ile-Gln-Glu-Leu-Arg-Pro-Thr-Leu-Asn-Glu-Leu-Gly-Ile-Ser-Thr-

T-11

,T-12 I

95

100

CNBr 2

I

105 Pro-Glu-Glu-Leu-Gly-Leu-Asp-Lys-Val CNBr 2

109 -

ST8 Fig.

1.

analyses of amino

Hz. I I

The amino acid sequence of the heme 5 subunit of bovine heart cytochrome oxidase. peptides obtained The symbols CNBr-, T-, and ST- represent from cleavage of the carboxymethylcysteinyl-derivative of the protein by cynogenbromide, trpsin, and succinylation, respectively.

(11, 12), methods used for the determination acids (13) have been described previously

1 Abbreviations:

Hz, hydrazinolysis;

Pth,

1015

of (14,

the Pth' 15).

3-phenyl-2-thiohydantoin.

derivatives

Vol. 76, No. 4, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Oxidase Hb S-chain Oxidase

20 --Val-Thr-Tyr-Phe-Asn@Pro-&-Ile-&aTrp-Glu

Hb S-chain

Gly-Lys-Val-Asn-Val-Asp-Glu-Val-Gly-Gly-Glu-Ala-Le~30

40

Oxidase

Lys-Gly-Met-Asn-Thr-Leu-Val-*-Tyr-Asp-Leu-Val--

Hb S-chain

Leu-Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe-Phe-

--

50 Oxidase

Pro-Glu-Pro-e-Ile-*-Asp-Ala@Leu-Arg@@&- -

Hb S-chain

Glu-Ser-Phe-Gly-Asp-Leu-Ser-Thr-Pro-Asp-Ala-Val-Met-Gly-

Oxidase

Arg-Leu-Asn-a-Phe --

70 --Glu-Val-

Hb $-chain

Asn-Pro-Lys-Val-Lys

Gly-Ala-

60

80 Oxidase Hb S-chain.

Gly-Leu-Ala-His-Leu-Asp-Asn-Leu-Lys-Gly-Thr90

Oxidase Phe-Ala-Thr-Leu-Ser-Glu-Leu-His-Cys-Asp-Lys

Hb S-chain

100 Oxidase Hb S-chain Fig.

2.

Comparison of the amino acid sequence of the 11.6 K bovine heart cytochrome oxidase and S-subunit of hemoglobin. Identical sequences between the 11.6 K heme 5 subunit bovine heart cytochrome oxidase and the S-subunit of hemoglobin are squared off. Circled residues indicate sequences of the heme subunit which are identical with the S-chain of other animal species (16). Underlined residues are those with only one base difference per codon. + indicates identical amino acid replacements observed in the abnormal S-chain of human hemoglobin (17).

RESULTS The methods

used and the results

of the heme -a subunit of the methods

that

will were

obtained

be discussed used

for

elsewhere.

to determine

1016

the amino

the

sequence

Only

a brief

acid

sequence

determination discussion of the pro-

BIOCHEMICAL

Vol. 76, No. 4, 1977

tein

will

be given

cleaved

by trypsin

raphy.

All

lysine

of the lysine

residue

Beckman

890 Protein

lysine in Fig.

to a pure

A final

residues

aliquot

and then

1 by the

with

symbol

1.

was succinylated

ST and many were

Another

in the

the NH2-terminus of the

and two fragments

to block

the

These

were

CNBr peptides

E-NH* groups

fragments

after

the

The

aliquot

of the

sequenced

1.

from

portions

trypsin.

for

was run

acids

bromide

Only

Fig.

was

chromatog-

except

aliquot

50 amino

cyanogen

with

cleaved

Another

in Fig.

state.

cleaved

were

by paper

90 as shown in

first

as shown

and an aliquot

isolated

residues

and the

was cleaved

and isolated

were

sequenced.

determined

derivative

sequenced.

peptides

residue

were

Sequencer

were

was carboxymethylated

and arginine

peptides

the protein

obtained

tryptic

21 and the arginine

tryptic

protein

The protein

and the

essential

of

here.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

are

were

of the

indicated

purification

by paper

chromatography. The sequence compared globin

(cf. -

Fig.

as well

of the bovine

heart

cytochrome

2) especially

with

the sequence

as hemoglobins

from other

oxidase

animal

heme a subunit

of the

species

g-chain

is

of human hemo-

summarized

by Fasman

sequence

of the 11,600

(16). DISCUSSION The br ief

report

dalton

subunit

single

polypeptide

here

that

documents

of bovine chain

there

are

to note found

and 40,000 that

in

but

subunit

daltons.

positions

21,

can be easily

which

acids.

These 31, 46,

72,

all

are:

The heme 2 is

by sulfenyl

acid

1017

bound

cannot

chloride.

be emphasized

to the

heme a and

weights it

is

readily

tightly remove

which

33; lysine

and the histidine very

of

interesting

heme ligands

55; methionine

and 108;

as the

should

ligands,

of the known

79

exists

have molecular

cysteine

74,

It

correspond

the potential

contains

trifluoroacetic

extracted

109 amino

Concerning

acid

The protein

The two subunits

2, 5, and 78. and even

amino

oxidase.

subunits

oxidase.

heme proteins.

dues on positions lated

and contains

the heme 2 subunit

in other

residues

cytochrome

two distinct

heme zz3 of cytochrome 11,600

heart

the complete

resi-

to the isothe heme 5

are

Vol. 76, No. 4, 1977

BIOCHEMICAL

The striking is

that

it

shows about

have been there

sequenced

in the

ancestor

for

91,

amino the

globin present

subunit

codon.

with

have

arisen

ly indicates

a homology

protoporphyrin

However,

the

although (cf. -

follows

it

is

of hemoglobins

which

is

heart

indicates

there

are

of 11.6 that

a sequence

it

indeed,

is

of hemo-

homology (18).

B-chain

visual

of the Efforts

K dalton

of hemo-

inspection in

in hemoglobin

subunit

certain-

the size

and the

becomes

a heme 5 rather

acid

the 11.6

and the

homology K dalton

at residues

S-chain

the differences

present

common

that

and the

However,

the observed

function

a possible

and myoglobin

oxidase

for

or pyrimidine

the same amino

to show that,

one allows

In addition,

to note

is

of hemoglobin

2).

one purine

contain

a common ancestor.

possible

Fig.

involve

(17)

cytochrome

and if

(see

interesting

that

programs

respectively, definitive

B-subunit

the heme 5 subunit

IX and heme a, which

heme 2 subunit,

the

which

It

the u-subunit

from

subunit

The homology

when

also

of bovine

of the oxidase

hemoglobins

observed It

sequence

species

replacements

to use computer

heme 5 subunit

animal

RESEARCH COMMUNICATIONS

acid

with

two hemoproteins.

compared.

are underway

unit

acid acid

as are

are

fined

20% homology

94 and 95, abnormal

replacements

globin

of the amino

from various

are many amino

change

14,

feature

AND BlOPHYSlCAl

of

oxidase

even more remarkable. is

not

than

clearly

de-

the heme ~3 sub-

6).

ACKNOWLEDGMENTS This HL 12576 Science

research from

was supported

the National

Foundation,

in part

Institutes

the American

Heart

from

of Health, Association

Grants

GM 22556,

GB 43448

from

GM 16767,

and

the National

and the Hawaii

Heart

Association. REFERENCES 1. 2. 3. 4. 5.

Kuboyama, M., Yong, F. C., and King, T. E. (1972). 2. 6375-6383. Yamamoto, T., and Orii, Y. (1974). 2. Biochem. (Tokyo) Phan, S. H., and Mahler, H. R. (1976). J. --Biol. Chem. Briggs, M., Kamp, P-F., Robinson, N. C., and Capiladi, Biochemistry It, 5123-5128. Tanaka, M., Haniu, M., Yasunobu, K. T., Yu, C. A., Yu, Biochem. Biophys. (1976). --Res. Commun. 6&, 357-367.

1018

Biol. --

Chem. 247,

75, 1081-1089. 251, 270-276. R. A. (1975). L.,

and King,

T. E.

Vol. 76, No. 4, 1977

6. 7. a. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Yu, C. A., Yu, L., and King, T. E. (1977). Biochem. Biophys. --Res. Commun. 3, 670-676. Crestfield, A. M., Moore, S., and Stein, W. H. (1963). J. --Biol. Chem. 238, 622-627. Klotz, I. M. (1967). Methods Enzymol. 11, 576-580. Spackman, D. H., Moore, S., and Stein, W. H. (1958). --Anal. Chem. 30, 1190 Edman, P., and Begg, G. (1967). Eur. J. Biochem. 1, 80. Bradbury, J. H. (1958). Bi0chem.T a, 475-482. Ambler, R. B. (1967). Memna-pal 2, 436-445. Pisano, .I. J., and Bronzert, T. J. (1969). J. --Biol. Chem. 244, 5597-5607. Tanaka, M., Haniu, M., Yasunobu, K. T., Evans, M. C. W., and Rao, K. K. (1975) Biochemistry &, 1938-1943. Tanaka. M., Haniu, M., Yasunobu, K. T., and Norton, T. R. (1977). Biochemistry 16, 204-208. Fasman, G. D. (ed). Handbook of Biochemistry and Molecular Biology, Vol. III, pp. 448-455. Hunt, L. T., Sochard, M. R., and Dayhoff, M. 0. in Dayhoff, M.O. (ed) (1972) Atlas of Protein Sequence and Structure, Vol. 5, pp. 67-87. Dayhoff, M. O., Hunt, L. T., McLaughlin, P. J., and Barker, W. C. in Dayhoff, M. 0. (ed) (1972) Atlas of Protein Sequence and Structure, Vol. 5, pp. D51-D85.

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