The role of tyrosine in the hemerythrin active site

Vol. 47, No. 2, 1972

THE

BIOCHEMICAL

ROLE

OF

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TYROSINE

IN

THE

HEMERYTHRIN

ACTIVE

SITE

:x* C.

C.

University

March

Received

Fan*

and

J.

Lyndal

York

Department of Biochemistry of Arkansas Medical School Little Rock, Arkansas 72201

13, 19’72 SUMMARY

Results presented here show that all lysine and three of the five tyrosine residues can be modified in hemerythrin without any alteration in the spectrum characteristic of the iron binding site. The two remaining tyrosines become available to modification when the iron is removed suggesting they are chelated to iron. Modification of the three unchelated tyrosines results in dissociation of the octomer into monomers suggesting a role of these tyrosines in the subunit binding.

In ing

a recent

that not

port

protein

sines, but

did

reagent.

of

was the

=W

Present delphia To

did

react

at the

not

in

were

In these

studies by

tyrosine center

address: 19140.

whom

inquiries

01972

by Academic

1971)

reacted site

could

not

tetranitromethane

of the

Dept.

should

hemerythrin.

Biochemistry,

addressed.

472 Copyright

Press, Inc.

hemerythrin,

an

The

iron

it or

To

Temple

oxygen

native

tyropro-

suggested

thatthe

inaccessible determine

of

trans-

two

the

was

were

unequivocally

that

and

remaining in

because

overlapped

be

to

indicat-

tetranitromethane

consequently,

ligands

residues

of

with

evidence

tetranitromethane

protein;

either

presented

brachiopods.

with

free

we

quickly

active and

we

the

York,

react

iron

tyrosines

active

67

sipunculids

109,

nitrated

and and

iron

the

modified

absorbing

*

to

in

two

site

16,70

ligands

8 and

latter

(Fan

tyrosines

were

tein

report

if

the

absorption

the

much

circumvent

Medical

to the

active

spectrum less

this

School,

the

strongly problem

Phila-

Vol. 47, No. 2, 1972 we

have

Since the

BIOCHEMICAL

utilized no

new

N-acetyl

imidazole

chromophores

hemerythrin

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

active

site

to

are

produced

can

be

modify

the

by

followed

this as

free

tyrosine

reagent

residues.

the

a function

of

spectrum

of

tyrosine

modi-

fication. For they

these

would

experiments not

amidinated

compete

protein

tyrosine

residues.

tyrosine

in

ing

modification

from

the

subunit

with

was

eleven the

reacted

This

approach

dissociation

lysine

tyrosine with

for acetyl

would phenomena

of unprotected

residues

lysine

the

were acetyl

imidazole allow

an

independent by

acetyl

first

modified

imidazole

then

to modify evaluation

of

the

free

the

role

of dissociation

so the

of aris-

imidazole.

METHODS Hemerythrin used in these experiments was crystalline oxygenated material prepared as previously described (Florkin, 1933 and Klop, 1957) and having an iron content of 0.81% and a E of 3400 cm-l-moleper M iron at 330 mu. The lysine residues were amidinated with ethyl acetimidate hydrochloride prepared according to McElvain and Nelson, 1942. A fresh working solution of ethyl acetimidate was prepared by dissolving the solid in water and neutralizing with 2N NaOH to pH 9.0. The protein was amidinated by adding the ethyl acetimidate at 2 hour intervals to a solution initially 3q7, in oxyhemerythrin buffered to pH 9 in 0.1 N borate. Generally four additions of a 100 fold molar excess acetimidate to protein was required to achieve 95- 100% conversion of lysine. The details were analogous to those of Wolfsy and Singer (1965). All reactions were performed at 0’. Excess reagent and hydrolysis products were remoded by dialysis against 0. 1 M Tris-chloride, pH 8.3. The number of free lysine residues remaining after each addition of acetimidate were assayed by the trinitrobenzene sulfonic acid method previously described (Fan and York, 1969). Amidinated oxyhemerythrin was acetylated with dry, crystalline Nacetyl imidazole (Eastman). The acetylation was performed at O” on a 2% solution of amidinated hemerythrin at pH 7.5, 0.05 M in sodium borate and Tris-chloride. A 200 fold molar excess of acetylimidazole to hemerythrin tyrosine was added at 2 hour intervals. In general four additions were required for the number of tyrosines reacting to remain constant. Acetylimidazole and its hydrolysis products were removed by dialysis against the pH 7.5 mixed buffer before evaluation of the extent of 0-acetylation was attempted. The number of unmodified tyrosine residues were evaluated by their reaction with tetranitromethane by measuring the nitrotyrosine chromophore absorption at 428 nm under conditions previously described (York and Fan, 1971) with the exception that the nitration was performed at pH 7.5 to prevent base catalyzed deacylation of the acetylated hemerythrin. After the nitration. was complete the excess tetranitromethane was dialyzed out at pH 7.5 and 0 then the pH was adjusted to 8.8 for the spectral determination of the amount of nitrotyrosine formed. The amount 473

Vol. 47, No. 2, 1972

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of 0-acetylation was independently checked by the decrease of 278 nm absorption upon formation of the 0-acetyl tyrosine. The absorbance was determined after dialyzing out excess reagents and their by-products. The AC was taken as 1160 (Riordan, Wacker and Vallee, 1965). s he percent denaturation of hemerythrin was determined after each modification by measuring the amount of iron which became soluble divided by the iron known to be protein bound originally. No attempt was made to remove soluble denatured material. Recoveries of 96 and 86 percent for amidinated and acetylated amidinated material was found. Sedimentation coefficients were determined by centrifuging 0.2 to 0.4% hemerythrin solutions in 0. 1 M Tris-chloride, pH 8.3 in a double sector cell at 59, 780 rpm in the Spinco Model E ultracentrifuge RESULTS All

the lysine

residues

AND

DISCUSSION

can be modified

by amidination

without

changing

spectra of oxyhemerythrin (-----) 1 mg/ml, OFigure 1. Absorption acetylated amidinated hemerythrin (-.-n-e) 1 mg/ml, 0-acetylated amidiand tetranitromethane treated nated hemerythrin k * - * * - * ) 1.95 mg/ml, ) 1.05 mg/ml. Spectra of 0-acetylated amidinated hemerythrin (oxy- and amidinated oxyhemrythrin were taken in 0. 1 M Tris-chloride, pH 8.3. Spectra of the acetylated samples were taken in 0.05 M Trisborate pH 7.5. the spectrum

that

is characteristic

of the iron

shows

a typical

oxygenated

hemerythrin

nation.

Within

experimental

error

was

successful

to the extent

binding

spectrum the spectra

of 95%. 474

Only

site.

before are

identical.

5% of the original

Figure and after

1 amidi-

Amidination TNBS

sensi-

BIOCHEMICAL

Vol. 47, No. 2, 1972

tive

lysine

residues

acid.

We

dine

renders It is

after

trast

to

it

protein

not

result

material value

is

mer

(Klotz

tiate

our

not

not

involved

can

the

decrease

ethyl conditions thrin.

be

of (Sigma) identical

278

pH

7.5

subunit

Figure

residues

nm

absorption.

would

not

to

0. 2%

that

used

It was undergo in

assay

new

buffer

475

now

group

conclude

reaction at 4.28 no

the

oxidato

Fig.

1. tetranitro-

nm

character-

unchelated

or found

measured

N-acetyl

by

tyrosine

tetranitromethane acetyl

hemerythrin the

be The

with

it was as

that by

in appears

that

found

residues

1965),

acetylated

until

octo-

amino

Independently

for

this

substan-

results

peak

amidinated

Tris-borate

native

spectrally

by

nitration the

for

tyrosine

suggesting

were

feel

the

site.

Klotz,

available.

0-acetylated SDS

1 no

on

does

We

the we

available

assayed

appeared

tyrosine

and

of what

was

were

and

binding

and

con-

charge

strongly

imidazole

formation

from

findings

ligands

N-acetyl

residues

of in

as

ami-

since

7.0.

reported

residues

(Keresztes-Nagy

seen

f .Z)

6.75

of the

modification

Denaturation at

the

absorption

(3.1

ester

the

with and

tyrosine

three

in

where

dissociation

w of 9’

These

site

Acetylation

of nitrotyrosine

that

80°

directly

tyrosine

As

the

In

1.

amidination

subunit a 525

an

oxygenated

Fig.

occurs,

lysine

binding

oxyhemerythrin

of the

the

its

1963),

with

to

acid.

spectrum,

in

from

that

chloride

the

nor

peak

lysine

maintained

1963).

Modification:

methemerythrin

unmodified

a single

iron

oxyhemerythrin

methane.

charge

sulfonic

of

sulfonic

dissociation

different

the

group

Keresztes-Nagy,

Keresztes-Nagy,

in

amidinated

extent

total

contention

trinitrobenzene

E amino

from

subunit

significantly

Tyrosine

istic

and

as

involved

of

(Klotz

in

and

are

deduced

a change

the

the

oxyhemerythrin

as

and

with

trinitrobenzene

that

altered

earlier

not

tion

note

sedimented

they

to

amidination

in

react of

unreactive to

is

to

conversion

succinylation

the

in

that

important

state

are

found

remained

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

under

amidinated

hemeryby

characteristic

heating

at

BIOCHEMICAL

Vol. 47, No. 2, 1972

hemerythrin

spectrum

residues

of

0-acetyl

tyrosine

in

The is

these

of

available

also

These

the

absorption

fication

1.96

cause

in

the

one(s)

had

these by

of

and of

and

tyrosine

only

three

that

this

modification

three

of the does

which

have

1963). tyrosines

is

(are)

buried

tyrosine

ligands. residues

involved

in

affect

however,

modiof the

octo-

S value

progress in

can

not

dissociation

is

residues

groups

does site;

1971).

because

a characteristic

Work

Fan,

of potential

active cause

and

iron

hypothetical

that

the

to subunit

of

evaluate binding.

ACKNOWLEDGMENT This tion

work

was

supported

by

a grant

from

the

National

Science

Founda-

(GB-30742X).

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.

Fan, C. C. and York, J. L. (1969) Biochem. Biophys. Res. Comm. 36, 365. Eorkin, M. (1933) Int. Physiol. 36, 247. --Arch. Keresztes-Nagy, S., and Klotz, I. (1965)Biochemistry 4, 919. H. A. (1957) Arch, Biochem. Klotz, I. M., Klotz, T. A., and Fiess, Biophys. 68, 284. S. (1963) Biochemistry 2, 445. Klotz, I. and Keresztes-Nagy, Riordan, J. F., Vallee. B. L. (1963) Biochemistry 4, 1460. Riordan, J. F., Wacker, W. E. C., Vallee, B. L. (1965) Biochemistry 4, 1758. Wofsy, L. and Singer, S. J. (1963) Biochemistry 2, 104. York, J. L. and Fan, C. C. (1971) Biochemistry 1659. - 10,

476

2.4

model

two

techniques

release

to

The

the

are

these

2.1

procedure.

(York

the

groups

subunits

this

present

render

and

show

Keresztes-Nagy, the

The

tyrosines

these

characteristic

monomeric

reactive.

earlier

two

and

iron

hemerythrin

three

of

represent

predicted

protein

loss

spectrum

(Klotz

which

the

appearance

by

therefore

that

conclusively

of these into

we

the

the

deacylated

tyrosines

possibility

unfold

results

0-acetylated

not

approachable

which

be

new

in

tetranitromethane was

which

experimentally

procedures

mer

two

the

resulted

were ester

hemerythrin

question not

which ethyl

of

ligands

disappeared

tyrosine

appearance

AND BIOPHYSICAL RESEARCH COMMUNICATIONS