Specific anion binding effects on the reactive sites of hemerythrin

Vol. 32, No. 2, 1968

BtOCHEMlCAL

AND B0PHYSlCAL

RESEARCH COMMUNICATIONS

SPECIFIC ANION BINDING EFFECTS ON THE REACTIVE SITES OF HEMERYTHRIN Dennis

W. Darnall,'

Biochemistry Northwestern ReceivedJune1'7,

the

of sipunculids,

is

atoms.

non-heme composed

et al., It

iron-containing of eight

essentially

The octameric

protein

reagents.

the

shown previously Fe sites

ligands enhances

as judged

(e.g. the

N3-,

rate

We have now found

produce

at

sulfhydryl

that

The effect

at

of experiments,

represented

from

on the

of

this

coordina-

sulfhydryl

the

behavior

sites

into

monomers.

anions

are

iron

locus,

and

iron

and

of both

bound

groups. Behavior

rates.

that

specific

different

(Groskopf

a variety

protein

into

and Klotz,

of octamer

certain

effect

with

Cl-),and

of dissociation

a site

a pronounced

complexes

of the

sub-

contains

residue

(Keresztes-Nagy

SCN-,

reactivity

by the

to hemerythrin,

form

identical

may be dissociated

1966), by mercaptan-blocking has been

carrier

1963) each of which one cysteine

anionic

M. Klotz

oxygen

contains

1965) that

types

and Irving

Department of Chemistry Evanston, Illinois

and Klotz,

each of which

subunits,

tion

Division,2 University,

(Keresztes-Nagy

two iron

Garbett

1968

Hemerythrin,

units,

Keith

Typical

the

iron

locus

Site

has been

one involving

coordination

by the

at Iron --w

equilibria

evident

equilibria,

from the

of hemerythrin

two

other may be

equation

'Postdoctoral Fellow of the National Institute of General Medical Sciences, U. S. Public Health Service, 1966-1968. 2This investigation was supported in part by a grant (~~-08299) from the National Heart Institute, U. S. Public Health Service. 264

Vol. 32, No, 2, 1968

BIOCHEMICAL

[HrFe111(He0)m18 + 8n~-

locus.

complex creates a net negative

make the Fe-ligand

charge at the iron

thermodynamically,

effects , and thus will

rium in equation (1) towards the aquo side. tard the rate of formation

The formation

near the iron should tend to

complex less stable,

through electrostatic

+ 8m He0

(e.g. OH'-, Ns-, NCS-).

Binding of a non-ligand

effects

RESEARCH COMMUNICATIONS

= [HrFes111(I,)n]8n-

where I, is the iron ligand of the ligand

AND BlOPHYSlCAL

of the Fe-ligand

drive the equilibIt should also reform.

Both of these

have been observed.

mo

P

3

I 350

I 400

0

A (m/d

Figure 1. Absorption spectra showing conversion of met-hydroxohemerythrin to met-aquohemerythrin in the presence of sodium perchlorate at 5°C. 1. 2. 3. 4.

2..

pH pH pH pH pH

8.62, 8.62, 8.62, 8.62, 8.62, 6.36,

0.00 3.75 1.00 3.75 0.00 2.50

M NaC104 x lo'* M NaClO4 x lo-= M NaC104 x 10-e M NaC104 xM 10e2 NaClO4 M NaC104

265

Vol.

32,‘No. 2, 1968

BIOCHEMICAL AND 8lOPHYSlCAL

Non-ligand anion binding nating anions to a solution [HrFealll(OH-)n]s,

RESEARCH COMMUNICATIONS

was detected by adding non-uoordiof met-hydroxohemerythrin

at pH 8.5.

The magnitude of the spectral

change (see Figure 1) towards met-aquohemerythrin, [HrFezlI1 (H20)mIsr then indicated anion binding binding

present.

Using this

as a criterion

we find that strong binding

with nitrate

ions,

ing with sulphate, ions.

the magnitude of non-ligand

weak binding

occurs with perchlorate

with phosphate ion and no bind-

anion binding

for certain

site

appears, therefore,

to

small anlons.

To ensure that the observed effects nation to the iron,

and

dodecylsulphate , acetate or trlchloroacetate

The non-ligand

be specific

for anion

the circular

dichroic

were not due to coordispectra of these solu-

Figure 2. Circular dichroic spectra of met-aquohemerythrin met-hydroxohemerythrin mixtures. ---------pH

7.0, 875 met-aquo

-*-pH

8.5, 94% met-hydroxo

---------pH

8.5,

0.091 M NaClO+, 93% met-aquo

Spectra recorded on Durrum-Jasco Recording Spectropolarimeter at 25'C in 0.1 M tris buffer adjusted to pH with solid cacodylic acid. 266

and

Vol.

32, No. 2, 1968

tions

BlOCHEMlCAL

AND B0PHYSlCAL

RESEARCH COMMUNICATIONS

were measured and compared with that of met-aquohemeryth-

rin at pH 7.0.

These are given in Figure 2 where perchlorate

the added ion.

The presence of about 0.1 M sodium perchlorate

alters

the circular

hemerythrin

dichroic

sensitive

spectrum from that of met-hydroxo-

to one which is essentially

met-aquohemerythrin.

Since circular

of perchlorate hemerythrin tration

studies

observed:

equilibrium

at a site equilibrium results

For a protein

pK values have been

half

of nitrate

a 20-fold

binding

that of perchlorate.

Ion bindlng

on this Similar

equilibria.

on the rate of forma-

species Is shown in Figure 3.

excess of sodium perchlorate

on

binds Clod- strongly

have been observed for other aquo-ligand

of a ligand

It is

has a pronounced effect

The effect

of perchlorate

concen-

1 x 10m3 M NaC104, pK 8.70.

and hence that hemerythrin

is approximately

The effect tion

on the aquo-hydroxo-

1 x 10m4 M NaC104, pK 8.28;

of perchlorate

near the iron.

out on the effect

the following

0.00 M NaC104, pK 7.83;

2.5 x 1O-4 M NaC104, pK 8.39; this

ion binding

(see Figure 1) equilibrium.

clear that the binding

complex.

have been carried

of 3 x 10e4 M in Iron,

is particularly

Is met-aquohemerythrin,

coordinational

and of nitrate

to that of

is clear that the species

formed in the presence of perchlorate Quantitative

identical dichroism

to*changes at the lron,it

and not an iron-perchlorate

is

The presence of

over protein

Iron concen-

tration

causes a large decrease in the rate of formation of the This phenomenon met-azidehemerythrln from met-aquohemerythrin. too clearly

demonstrates that perchlorate

ion is bound near the

iron in this protein. The techniques ligand

described do not permit us to see whether

anions that can be coordinated

bound to the specific

anion binding 267

to the iron are also sit?e. However electrophore-

Vol. 32, No. 2, 1968

BlOCHEMlCAL

0.5

AND B~OPHYSICAL RESEARCH COMMUNICATIONS

I

I

I

3 fn 0.4 -

! a

0.2

I IO MlNUlf5

1 5

0

Figure 3. Rate of formation aquohemerythrin.

1 15

20

of met-azidehemerythrin

A.00

M NaC104

---------8.35

x 10D3 M NaC104

from met-

Both solutions contained 3.46 x 10e4 M Fe, 5.25 x 10e4 M NaN3; pH 7.0, 5°C.

tic

work (Keresztes-Nagy

indicates native

that chloride

and Klotz,

I.M.,

unpublished

ion is bound to oxyhemerythrin

results) in its

state. Behavior at Sulfhydryl Not only do Clod- and NOs- affect

these ions also simultaneously reaction

Site the iron locus,

"mask" the -SH group in its

with mercurials.

This was demonstrated by the following stoichiometric cysteine

but

amount of salyrganic

residues,

met-aquohemerythrin was dissolved,

acid,

in relation

was added to a solution at pH 7.0.

the solution

Immediately

was divided

268

experiment. of (7.0 after

A to

x low4 M Fe) the mercurial

into two portions.

To

Vol. 32, No. 2, 1968

BIOCHEMICAL

- 9.1 - 0.0

Ww Lower

x lo-* M NaCl04 M NaC104

one portion

sodium

perchlorate

tion

x lo-*

M.

of 9.1

tris-cacodylate same for

the

samples

both were

Analytical

whereas

sociated

octameric

with

protein

essentially

complete

found

when the

concentration

of the

single

cysteine

perchlorate for

binding

in binding C104-

the

sedimented evident

was about

that

the

in

protein.

strongly

of the

269

only

subunit)provides the

Model sam-

2s

non-disexperi-

sulfhydryl

indicates

4'C,

4 the (into

was the

and producing

observed.

Spinco

In other

This

(per

at

in Figure

of perchlorate

site

the

showed

of

strengths

overnight

particles).

protection

the

same amount

80% dissociated

perchlorate (6s

concentra-

ionic

standing

As is

ments

that

to keep

simultaneously

to a final

portion

After

solutions.

perchlorate

particles)

other

was added

Ultracentrifuge.

without

was added

To the

buffer

the

locus

RESEARCH COMMUNICATIONS

Sedimentation of met-aquohemerythrin. Both solutions 4. contained07.0 x lo-* M Fe, 3.5 x lo-* M salyrganic acid at Both diagrams from photographs taken 90 %&.$ zf&r reaching speed of 59,780 RPM.

Figure

ple

AND BlOPHYSlCAL

was same as that the

effects

a

E

Vol. 32, No. 2, 1968

BIOCHEMICAL

Other sedimentation nitrate

AND B~~OPHYSICAL RESEARCH COMMUNICATIONS

experiments,

as the -SH protective against dissociation

ate.

This lessened effectiveness parallels Titrations

its

out with

At pH 7 .O nitrate

agent.

tects tivity

were carried

but not as effectively

relative

pro-

as perchlor-

of NOe- with regard to -SH reac-

effectiveness

of the sulfhydryl

at the iron site.

group with p-chloromercuri-

benzoate by the method of Boyer (1954) have also shown markedly reduced rates of reaction extensive

in the presence of Clod-.

-SH titration

studies

where (S. Keresztes-Nagy, The attenuating reactivity

are currently

private

effects

communication).

contrast

and Klotz,

anions on -SH

to the marked enhance-

ments in rates produced by iron-coordinating azide (Keresztes-Nagy

in progress else-

of these non-ligand

stand in striking

Much more

ligands

such as

1965).

Discussion These observations

Indicate

that hemerythrin

and a few other ions very strongly ordinated kinetic

to the Fe atoms. and equilibrium

electrostatic also affects Interaction

The non-ligand

repulsive

interactions.

the reactivity

strongly

anion

However, this for it has also

The stoichlometry that the effects

C104- bound at a single

ClOh- ion affects

the SH locus we have herewith site

the

with uncharged N-ethylmaleimide,

of C104- indicates

are produced by a single

binding

The non-ligand

may not be wholly electrostatic

Since this

being co-

anion affects

of the SH group.

as well as with anionic mercurials.

subunit).

their

behavior at the Fe locus, probably by

been observed in titrations effects

without

binds Clod-

and the single

site

of the described (on each

both the Fe locus and

strong evidence that the oxygencysteine

in close proximity. 270

residue in hemerythrin

are

Vol. 32, No. 2, 1968

8lOCHEMlCAL

AND E~OPHYSICAL RESEARCH COMMUNICATIONS

References Boyer,

P.D.,

J. Am. Chem. 5, ---

76,

4331

Grosk~PP~,r.B~~~h~~:~~~~,Jj7~g,(E;l~~%pliash, Y Keresztes-Nagy, (1963)

Keres&z;yagg,

(1954)

E.,

and Klotz,

S.,

and Klotz,

I.

M.,

Biochemistry

Z?

923

S .,

and Klotz,

I.

M.,

Biochemistirg

4,

919

l

271