Spin state equilibria in soybean ferric leghemoglobin and its complexes with formate and acetate

Vol. 88, No. 2, 1979

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

BlOCHEMlCAL

May 28, 1979

Pages

713-721

SPIN STATE EQUILIBRIA IN SOYBEAN FERRIC LEGHEMOGLOBIN AND ITS COMPLEXES WITH FORMATE AND ACETATE

School

J. Trewhella and P.E. Wright,* of Chemistry, University of Sydney, N.S.W. 2006, Australia and

C.A. Appleby, Division of Plant Industry, Commonwealth Scientific and Industrial Research Organisation, Canberra, A.C.T. 2601, Australia Received

April

7, 1979 SUMMARY

The interactions of fluoride, acetate and formate with soybean ferric leghemoglobin &have been investigated by ‘H NMR spectroscopy. In the presence of fluoride or acetate leghemoglobin is locked into a high spin ferric conformation whilst the formate complex exists as an equilibrium Both formate and acetate ligate mixture of high and low spin states. directly to the iron and the different magnetic properties of the complexes are attributed to steric constraints within the heme pocket. Leghemoglobins transport

structure

2.8 .8 resolution

acids

(3)

brium

mixture

molecular

show that differences

* Author

(I),

of spin

basis

for

including

states these

in the

and other

and for-mate iron

ferric

spin

to whom correspondence

(6).

protein

properties

fluoride,

state

iron

in these

should

nodules

(2).

long

to exist to gain

chain

an insight

on the interaction

acetate ligands

and formate. and discuss

carboxylate

to

Despite in many carboxylic

as an equili-

we have undertaken here

(1).

determined

from myoglobin

to bind

In order

are both

has been

differs

We report

a with

lupin

in oxygen

root

of myoglobin

ability

(4,5).

leghemoglobin

acetate

to that

its

of the

of leghemoglobin

ferric

similar

function

legume

from

leghemoglobin

and the tendency

NMR studies soybean

of leghemoglobin

resemblance,

properties

which

nitrogen-fixing

and is very

structural

of its

hemeproteins

in Rhizobium-infected,

The tertiary

this

are monomeric

into

the

detailed of We unexpected

complexes.

be addressed. 0006-291X/79/100713-09$01.00/0

713

Copyright All righrs

@ 1979

by Academic. Press. Inc. in anyformreserved.

ofreproducrion

BIOCHEMICAL

Vol. 88, No. 2, 1979

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

MATERIALS

AND METHODS

Soybean ferric leghemoglobin a was purified by a previously described procedure (7). For NMR measurements the protein was repeatedly dialysed against phosphate buffer (lOmM, pH 7.0) in D20. The carboxylate complexes were prepared by addition of sodium deuteroacetate (28-fold excess) or sodium formate (lo-fold excess) to ferric leghemoglobin. The pH was adjusted to 6.5 by careful addition of DC1 or NaOD. Proton NMR spectra were recorded using a Bruker HX-270 spectrometer. Dioxan was used as an internal standard but all peaks are referred to TSS (trimethylsilylpropanesulphonic acid). Optical difference spectra were recorded using a Cary 14 spect rophot omet er . RESULTS AND DISCUSSION The NMR spectrum shown in fig.

of ferric The absence

l(a),

resonances

has been

broadening

due to relatively

and low spin heme iron ration

which

Their

broadness

relaxation other

is

characteristic

resolved

binds

(4,s).

In the

into

resonate

hemoglobin

group

between

Ligation

high

of fluoride

high

spin

ferric

configu-

group

protons.

slow

iron

fluoride

unpaired

complex

spectrum

electron

spin

of leghemoglobin

has been reported

spin to the

from the heme methyl

in the

deuteroacetate been

of leghemoglobin

and

for

confirmed

iron for

like

configuration. as those

in which

myoglobin

clearly

atom in leghemoglobin. the acetate

714

a high

complex

spin

well62.9,

61.0

fluoride,

abnormal group

indicates This of lupin

is

group

B-subunits ligated

direct

and locks

The heme methyl

of the

a carboxylate

Thus the NMR spectrum to the

at 65.9,

deuteroacetate,

ferric

same region

to form

deuteroacetate

are observed

Clearly

spin

(10)

leghemoglobin

resonances

l(c)).

a high

M Milwaukee

atom (11).

recently

to

50 ppm

to ferric

spectrum

ppm at 30°C (fig.

protons

attributed

near

A similar

reversibly

heme methyl

leghemoglobin

iron

(8).

group

of resonances

to the of the

loss-‘)

a fully

set

and is

is

(9).

Acetate complex

into

arise

attributed

(ca. -

temperatures.

region

heme methyl

(6)

exchange

to a broad

probably

hemeproteins

fluoride

slow

rise

low field

of hyperfine-shifted

leghemoglobin

(5) and gives

in the

by us previously

at ambient

atom locks

l(b))

54.9

reported

states

(fig.

leghemoglobin

of

to the

ligation

conclusion leghemoglobin

of

has by

Vol. 88, No. 2, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

L L

a

A.-

I

I

80

I

I

60

,

I

40

.L

__-I---

20

0

PPm Figure

X-ray

1.

270 MHz proton NMR spectra at 25’C of (a) ferric soybean leghemoglobin a (2.6 mM, pH 7.0). (b) Leghemoglobin fluoride (42 mM fluoride, pH 6.5). (c) Leghemoglobin deuteroacetate (27 mM deuteroacetate, pH 6.5).

diffraction

(12).

hy-perfine-shifted deuteroacetate exhibit

a normal

is

The observed

heme methyl

group

in

the high

spin

Curie

law

(l/T)

temperature resonances

ferric temperature

715

dependence confirms

state

(fig.

dependence

that 2).

of the leghemoglobin The resonances

and shift

upfield

Vol. 88, No. 2, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

716

Vol. 88, No. 2, 1979

BlOCHEMlCAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

1

620

AA

T

=O.l

1

, ,

/

493 1 ,--

/

, -\

\

,’ 3’

\

, \

, ‘\

540 1 . -’

I

I

I

,

I

/

,

I

,

,-

\

i

,

S

Ferric

Lba

’

minus

Ferric

Acetate Lba

\

I

‘__.-___--

,,,’

Ferric

Lba

minus

Ferric

Formate Lba

T

631 450

500

600

550

650

700

Optical difference spectra for acetate and formate complexes of soybean leghemoglobin (50 uM in 50 mMMES/NaOHbuffer, pH 5.30 at 20°C). (a) Spectrum of leghemoglobin acetate (total acetate concentration is 250 mM)versus leghemoglobin. (b) Spectrum of leghemoglobin for-mate (total formate concentration is 250 mM) versus leghemoglobin.

Figure 3.

towards the positions at which they would resonate in a diamagnetic hemeprotein

(ca. 3 ppm). -

It is clear from the optical interacts

difference

spectra of fig.

with leghemoglobin in a rather different

other straight

chain aliphatic

carboxylates).

peaks at 620 nm and 493 nm and the ferric

3 that formate

manner to acetate (and

The charge transfer

band

hemochrometrough at 540 nm are

in accord with formation of a high spin complex when ferric

leghemoglobin

combines with acetate at 20°C. On reaction with formate, however, the appearance of ferric

hemochromepeaks at 535 and 562 nm and charge transfer

band troughs at 502 and 637 nm in the difference ferric

spectrum suggests that

leghemoglobin adopts a more low spin structure.

717

Vol. 88, No. 2, 1979

Further

BIOCHEMICAL

information

leghemoglobin

resonances

the

heme methyl

four

higher

fields

than

the Curie

law since

ture,

i.e.,

other

two resonances

an increase

is

indication

increase

Similar

of the

slow

high

(13) .

spin

in marked

loss-‘)

Exchange

and low are

the origin

between scale.

spin

contrast

formate,

to shift

of the spin

leghemoglobin

state

equilibria.

in leghemoglobin

formate

probably

‘A and ‘T energy

states,

constrained

field nitrogen

produced

by formate,

donor

atoms.

We have previously leghemoglobin involves

motion

of the

and ferric

arises

from

to lie

in energy

crossing

is generally

from a protein distal

conformational

histidine

718

on and off

state

differences

in

interconversion

four

iron

between ligand

pyrrole very

exchange

change the

(~a.

by the

Such inter-system

spin

This

crossing

and the

the slower

the

rate

state

histidine

that

the

since

exchange

spin

of

leghemoglobin

temperature.

intersystem

close

complexes

is fast

and indicates

The rapid

to an

between

the proximal

suggested

arises

and imidazole

state (6)

of spin

conclusion.

changing

to the relativelyslowspin

in ferric

this

formate

with

This

leading

of interconversion

steadily

do

The temperature

with

acetate

of leghemoglobin

than

mixture

species.

the azide

The

temperature.

in temperature

in accord

The rate

states

observed

observed

is

have been made for

on the NMR time

resonances is

spectrum

4).

Such behaviour

increasing

spin

follow

tempera-

dependence

acetate.

an increase

does not

(fig.

of an equilibrium

of the high

optical

observations

myoglobin is

formate,

in the proportion

dependence

positions

with

of leg-

increasing

temperature

shift

of the existence

in leghemoglobin

with

from

at considerably

dependence

of leghemoglobin

of

well-

in the spectrum

downfield

much smaller

in hyperfine

exhibits

are observed

diamagnetic

complex

ppm at 30°C arising

temperature

two of them shift

for-mate

which

resonances

their

resonances

reflects

These

corresponding

exhibit

of the

and 38.6

4).

move away from their

the corresponding

39.3

(fig.

Further,

state

NMR spectrum

43.3,

groups the

acetate.

states

from the

at 45.1,

hemoglobin

clear

on the magnetic

was obtained

resolved

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

rapid in ferric

and probably atom (6).

(14).

Vol. 88, No. 2, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 88, No. 2, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Why does the formate complex of leghemoglobin exist as an equilibrium mixture of high spin and low spin states at ambient temperatures whilst the acetate complex is fully

high spin? This behaviour is unique to

leghemoglobin, myoglobin fox-mate for example being a high spin ferric complex with NMRspectrum similar to that of leghemoglobin deuteroacetate The inherent ligand field

strengths of formate and acetate ligands are

expected to be very similar, globin should exhibit

(9).

in which case their

complexes with leghemo-

similar magnetic properties.

The observed differences

in leghemoglobin formate and leghemoglobin acetate can, however, be rationalised

if it is assumedthat steric

pocket interfere

constraints

with the binding of the bulkier

to a weaker ligand field this interpretation,

within the heme

acetate ligand,

and a high spin ferric

complex.

fonnate binds more strongly

leading

In support of

to ferric

leghemoglobin

= 0.23 mM, pH 5.3 and 20°C) than does acetate (Kdiss = 4.5 mM, (Kdiss pH 5.3 and 20°C) (15), which has an affinity for leghemoglobin similar to that of longer straight a-CHa group of aliphatic affinity

chain carboxylic carboxylic

for leghemoglobin.

acids (3).

acids substantially

Isobutyric

Substitution

at the

reduces their

acid binds only weakly to ferric

leghemoglobin (Kdiss ~a. 500 mMat pH 5.3 and ZO'C, as determined by the spectrophotometric to bind at all.

method described in (16))and lactic

acid appears not

Thus it appears that whilst the hemepocket of ferric

leghemoglobin is more open than that of ferric

myoglobin, allowing the

binding of long chain carboxylic

constraints

pocket are still

acids, steric

important and influence

within the

the nature of the metal-ligand

bond.

ACKNOWLEDGEMENTS We thank Mrs J. Wellington the preparation for financial

and Mrs L. Grinvalds for assistance with

of leghemoglobin and the Australian support and for the use of facilities

720

Research Grants Committee at the National NMRcentre.

Vol. 88, No. 2, 1979

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

REFERENCES 1.

2.

3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

Appleby, C.A. (1974): The Biology of Nitrogen Fixation (Quispel, A. ed.) pp, 521-554, North-Holland Publishing Co., Amsterdam. Vainshtein, B.K., Arutyunyan, E.G., Kuranova, I.P., Borisov, V.V., Sosfenov, N.I., Pavlovskii, A.G.. Srebenko, A.I., Konareva, N.V. and Nekrasov, Yu. V. (1977) Dokl. Akad. Nauk. SSSR233, 238-241 (English Edition: 233, 67). Ellfolk, N. (1961) Acta Chem. Stand. 15, 975-984. Ehrenberg, A. and Ellfolk, N. (1963) Acta Chem. Stand. II, S343-S347. Appleby, C.A., Blumberg, W.E., Peisach, J., Wittenberg, B.A. and Wittenberg, J.B. (1976) J. Biol. Chem. 251, 6090-6096. Wright, P.E. and Appleby, C.A. (1977) FEBSLett. z, 61-66. Appleby, C.A., Nicola, N.A., Hurrell, J.G.R. and Leach, S.J. (1975) Biochemistry 14, 4444-4450. Ollis, D.L., Eight, P.E., Pope, J. and Appleby, C.A., submitted for publication. Morishima, I., Neya, S., Ogawa, S. and Yonezawa, T. (1977) FEBSLett. 83, 148-150. Fung, L.W.-M., Minton, A.P., Lindstrom, T.R., Pisciotta, A.V. and Ho, C. (1977) Biochemistry 16, 1452-1462. Perutz, M.F., Pulsinelli, P.D. and Ranney, H.M. (1972) Nature New Biol. 237, 259-263. Vainshteln, B.K. and Arutyunyan, E. (1978) abstract, Int. Symp. Biomolecular Structure, Conformation, Function and Evolution, Madras, India. Morishima, I. and Iizuka, T. (1974) J. Amer. Chem. Sot. 96, 5279-5283. Binstead, R.A., Beattie, J.K., Dose, E.V., Tweedle, M.F. and Wilson, L.J. (1978) J. Amer. Chem. Sot. 100, 5609-5614. Appleby, C.A. and Wittenberg, J.B., manuscript in preparation. Appleby, C.A., Wittenberg, B.A. and Wittenberg, J.B. (1973) Proc. Natl. Acad. Sci. USA'0, 564-568.

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