Magnetic circular dichroism of non-heme iron proteins

Vol. 51, No. 4,1973

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

MAGNETICCIRCULARDICHROISMOF NON-HEME IRONPROTEINS* D. D. Ulmer', B. Holmquist and B. L. Vallee+ The Biophysics Research Laboratory, Department of Biological Chemistry, Harvard Medical School, Division of Medical Biology, Peter Bent Brigham Hospital, Boston, Massachusetts

Received

March

5,

1973

The magnetic circular dichroism (MCD) at 45 kgauss has been determined for a group of non-heme iron proteins. Both transferrin and conalbumin exhibit a single, positive ellipticity band at 330 nm ferre([@I = 560). Oxy- and methemerythrin, spinach and clostridial dox&s and rubredoxin all display distinctive multibanded spectra which may reflect such factors as coordination of the metal, its ligands, metal bridging by other atoms, and varying degrees of metalmetal coupling. The MCDspectra of both ferredoxins and rubredoxin undergo dramatic change upon oxidoreduction providing a potential means for relating the electronic structure of the iron to protein In contrast to the plant ferredoxins, the magnetic field function. does not significantly affect the CD spectra of adrenodoxin and putidaredoxin. Studies of the extrinsic

Cotton

effects

of metalloproteins

circular

dichroism (CD) have contributed

importantly

of their

conformations and metal-binding

sites

by

to the understanding

(l-3).

Whereas conventional

CD signals arise only from asymmetric chromophores, imposition of a properly oriented magnetic field,

MCD, can induce optical

Effect

-- in virtually

origin

of the Faraday Effect

distinct

all

electronic

transitions.

activity

-- the Faraday

Since the physical

and that of the Cotton Effect

are quite

(4,5) the former would be expected to provide further

into the electronic

structure

To assess the potential

insight

of the metal sites of metalloproteins. usefulness of magnetic circular

dichroism

@CD), we have examined a group of non-heme iron proteins which have been characterized

previously

by conventional

*

CD and resonance spectral methods.

This research was supported by Grants GM-15003 and GM-02123 from the National Institutes of Health of the Department of Health, Education and Welfare. tPresent address: Martin Luther King, Jr. Hospital, Department of Medicine, Los Angeles, California. + To whominquiries should be addressed. Copyright 0 I9 73 by Academic Press, Inc. AN rights of reproduction in any form reserved.

1054

BIOCHEMICAL

Vol. 51, No. 4,1973

A preliminary proteins

report

has been Metal

METHODS:

given

(Sigma

and measured

in

was diluted

work

of human serum

Chemical

to 1 mg/ml

in

0.15

a gift

Lovenberg,

a gift

of Dr.

to remove

pH 8.0, Dr.

'T. Kimura,

prior

ethanol

Drs.

in 0.05

M Tris-Cl,

Chemical

Co.)

several

days

to remove

MCD spectra a Varian

was dialyzed

were

Model

measured

of 40 to 45 kgauss. dispersion experimental MCD is NM for

conditions

expressed

is

calculated

appropriate

solvent

N2 prior

oxidase,

61

CD spectra absence

were

from butterfat

pH 8.5,

for

All

equipped

at field to permit

of a magnetic

CD.

instrument

measured

cm2 decimole

conventional

CD

solenoid

was adjusted

as [O]M in degrees as for

liquid

by

sulfate.

width

in the

under

of

just

prepared

M Tris-Cl,

on a Cary Model

The slit

a gift

pH 7.49,

generously

Xanthine

superconducting

of 2 nm or less.

phosphate,

0.01

Hemerythrin,

adrenodoxin,

was stored

versus

ammonium

V-4145

while

pH 7.4.

The

0.1 M Tris-cacodylate,

Putidaredoxin,

C. Gunsalus,

also

pH 7.3.

was 2.55.

against

M sodium

Lovenberg,

rubredoxin,

material

salt,

in 0.01

and I.

(Sigma

with

and excess

measurements.

J. L. Lipscomb

to measurement

nm of this

type to measure-

Walter

M Tris-Cl,

Co.)

previously

pH 7.3,prior

by Dr.

in 0.15

Chemical

ferredoxin,

and -~ Clostridial

was dialyzed

was dissolved

to spectral

provided

was dissolved

Klotz,

Spinach

M NaCl,

pH 7.28

at 2801490

Irving

substituted

as described

pH 8.5.

kindly

on 0.1 41 T&s-Cl,

of absorption

of Co(II)

(Hoechst

prepared

M Tris-0.8

ferredoxin,

of Dr.

studies

transferrin

were

buffer,

was dissolved

ratio

Co.)

0.1 M Tris-Cl

Clostridial

and related

RESEARCH COMMUNICATIONS

(6,7).

complexes

and conalbumin

ment.

of this

AND BlOPHYSiCAL

-1

under

strengths

spectral identical

field. kilogauss

samples

-1

were

, where

corrected

blanks.

RESULTS: Figure broad

negative

1 compares

the CD and

CD ellipticity

band

MCD

spectra

centered

1055

of iron

transferrin.

at 465 nm coincides

A precisely

(1) III,

Vol. 51, No. 4,1973

with

the Xmax of the absorption

band at the

BIOCHEMICAL

330 nm.

A magnetic

330 nm band but

not

spectrum; field

that

AND BIOPHYSJCAL RESEARCH COMMUNlCATiONS

there

is

1. CD spectra presence ( of transferrin ( text.

at 465 nm (Figure

1).

between

measurements

field

(Figure

1, insert),

([O],

= 560).

This

is

not

altered

though

temperature to that

2 a-c

and of a bacterial per

mole

(Figure shows

thought

are

similar,

very

exhibits

three

but distinct

the

om

presence

a B-term

in temperature

from

of a C-term

requires

of a magnetic

signal

at 330 nm

since

its

and +30"

the

intensity

C (See below),

examination

is virtually

of a wider identical

1, insert). of hemerythrin,

of the protein

(8).

MCD spectra

from

Hemerythrin

ferredoxin. identical

positive

-30

of conalbumin

to have nearly

their

and absence

represents

the MCD spectra

and a plant

and met-forms

enhances

The MCD spectrum,

sharp

The MCD spectrum

the oxy-

markedly

as a single,

likely

elimination

of transferrin

Figure

atoms

transition

range.

in the

manifests

by variations

definitive

positive

of human serum iron transferrin in the absence ( ..a* ) ) of a 47 kgauss magnetic field. INSERT: MCD spectra ) and conalbumin ( - - - ). For conditions see the

and

difference

a sharper,

of up to 47 kilogauss

WAVELENGTH,

Fig.

also

differ

MCD bands,

1056

contains

electronic The CD spectra significantly: at 330,

sipunculid

worms,

two iron

structures of the

in both two forms

oxyhemerythrin

380 and 503 nm, superimposed

Vol. 51, No. 4,1973

BIOCHEMKAL

AND EtOPHYSlCAl

RESEARCH COMMUNICATIONS

400 500 WAVELENGTH, nm

300

600

of a) oxyhemerythrin ( ) and methemerythrin Fig. 2. MCD spectra ( - - - ); of b) oxidized > and reduced ( - - - ) clostridial ( ferredoxin; and c) of oxidized ( ) and reduced ( - - - ) spinach ferredoxin. For conditions see text.

upon a broad (Figure Jith

envelope

2a).

of positive

In contrast,

a positive

ellipticity

methemerythrin

increasing generates

peak at 438 nm, and a negative

to be a highly

discriminative

sensor

of the

with

a broad

trough

at

electronic

decreasing biphasic

wavelengt

band

350 nm.

MCD appears

properties

of the

oxy-

and met-forms. A high absorption

magnetic

field

or conventional

(Figure

2 b),

plified

by the oxidized

ellipticity doxins

that

bands

over

MCD may serve

properties.

spinach and above

Both

yet

not

observed

Clostridial

(2 g at Fe/mole)

protein

which

those

transfer

additional

to correlate

details of oxidized

ferredoxin

in electron

resolves

resolves

CD spectra

and spinach

function

dithionite

also

seen in and their

transitions, their

the MCD and CD spectra

structural

seven

2c) as exemdistinct

the CD spectrum. reduction

with

(Figures with

of xanthine

1057

(8 g at Fe/mole)

(Figure

exhibits

in the

The

ferre-

sodium

Zb,c)

suggesting

pertinent oxidase

new

functional (9)

closely

Vol. 5 1, No. 4, 1973

resemble

those

analogous

further

Above

ferredoxin.

The implication

structures

of both

intense

bands.

390 nm, may represent particularly

these

RESEARCH COMMUNlCATfONS

non-heme

that

this

reflects

iron

proteins

study.

300 nm the MCD of oxidized

overlapping,

Reduction

AND BIOPHYSICAL

of spinach

electronic

requires

BIOCHEMICAL

markedly

A biphasic

an A-term

helpful

312 and 336 nm (Figure

3) (see

the environment

the negative

3) identified

manifests

component,

(Figure

in defining enhances

rubredoxin

as an envelope

centered

between

discussion), the single

and positive

ellipticity

previously

in

380-

and could

of

of

iron

be

atom. bands

the CD spectrum

at

of

rubredoxin.

of oxidized Fig. 3. MCD spectra For conditions see the text.

Adrenodoxin, strengths supplied

(Kimura, by Dr.

to 45 kilogauss, plant visible

in accord

I.

Gunsalus, indicating

ferredoxins, and near

DISCUSSION:

personal

both

with

these

ultraviolet

characteristic

) and reduced

earlier

observations

communication), do not that

Zeeman splitting

an atom generates

( -

iron

at lower

field

and putidaredoxin,

exhibit

in contrast

proteins

( - - - ) rubredoxin.

lack

appreciable

kindly

MCD at fields

to the more negative significant

degeneracy

up

potential in

the

transitions.

of degenerate MCD signals

1058

ground

or excited

categorized

states

in accord

of with

Vol. 51, No. 4, 1973

their

origins

and differentiated

dependencies. are

BIOCHEMICAL

C terms

temperature

by means of their

are monophasic

dependent,

the

phasic

and temperature-independent

mixing

of non-degenerate

dipole

polarization

characteristics

e.g.

present

is

as well

as the

As concerns

ligands

the

Transferrin octahedrally metals

coordinated

are not

complexes, in time

their

and conalbumin

thought

binding resonance

each of the

The virtually

two irons

identical,

prove

and CD spectra, of overlaps.

The

to simpler

systems,

compared

MCD spectra

doubtless

the valence

of the metal,

is

by other

coordination

atoms,

geometries.

of cobalt

suggesting

two high and phenolic

(12).

two iron

atoms

spin

II

those

complexes

of some

Fe (1II)atoms

oxygen

Based on the seems identical

have been

interpreted

may exist

in a different

single

absorption

the MCD spectra

contain

to interact

signals

should

molecules,

(6).

to nitrogen

of the

it

metal-bridging

geometries

metalloenzymes

of more complex

atoms and their

found

of particular

substituted

iron

interaction,

we have

calculations.

empirical.

including

iron

theoretical

or because

much more

of factors

latter,

in

magnetic

to spectroscopic

However,

though

of the non-heme

of the

for

probabilities

of metal-metal

to be characteristic Co(I1)

electric

and of polarization

analysis

concealed

remains

by a variety degrees

the

such expectations,

diversity

varying

are monoinduced

reflect

can lead

basis

in

transitions

interpretation

influenced

These

as yet uncertain.

support

The rich

which

of different

states

a quantitative

of low transition

studies

spectral

the former

field

molecules

electronic

transitions.

in delineating because

B terms,

states

chromophoric

MCD can assist

metalloproteins,

either

are biphasic;

from magnetic

and excited

of their

provide

to which

valuable

result

of simple

of their which

The degree

not.

and temperature

(5).

properties

assignments

are

RESEARCH COMMUNICATIONS

patterns

and A terms

latter

ground

The MCD spectra and symmetry

AND BIOPHYSICAL

transition

1059

in

ligands

(11).

stability

differences

that

conformation

the MCD spectra

The

of their

though

to suggest

(10)

at any given (13,14).

of the

two

Vol. 5 1, No. 4, 1973

proteins

BIOCHEMICAL

(Figure

to oxygen

1) may be characteristic

significantly

features

of all

of these

coupling

of their

metal

regard

positions that

the

transitions is

and oxidized

thought

of hemerythrin

their

(17)

to form

accounting

perhaps

for

the differences

in nearly

those

spectra.

indicate

may respond

between likely

In

the same spectral ferredoxin

proteins

might

degrees

distinctive

a bridge

and sulfur

electronic

with

Clostridial

in both

ferredoxins

varying

together

bands

iron

the

to reflect

for

ellipticity

and plant

However,

taken

account

of the

Oxygen

closely

play

features

to the adjacent

an analogous of their

role

MCD which

similar. The sulfur

three

positive

(Fig.

2 c).

bound

However,

ferromagnetic studies

both

the iron

that

investigation

spectrum other

apparent

additional

ultraviolet

coupling

between

forms

their

oxidized

of these

of these

of the oxidized non-heme A-term

iron

centered

noted maxima

in each.

proteins

from

distinguish anti-

by NMR

ferredoxins

It

above

strong

as suggested

and from

exhibit

differ

each other,

suggesting

may be anticipated

different

sources

may

variations.

of cysteine spin

centers,

and bacterial

environment

also

due to unusually

equivalents

Fe atom of oxidized

and high

and visible

metal

of plant

ferredoxin,

of the bands

perhaps

protein,

of a series

groups

spinach

the position

atom has a unique

The single

Fe(III)

of oxidized

at or near

the plant

from

the basis

sulfhydryl

atoms

The reduced

that

clarify

of

yet

exchange

(15).

strikingly

iron

MCD maxima

the MCD spectrum

the

16) which,

positive

in some ferredoxins,

spin

15,

2, a-c).

are believed

oxyhemerythrin

field. atoms

(Fig.

may likely

three

of both

magnetic

are

Fe (11% coordinated

spin

and the bacterial

proteins

(8,

ligands,

analogous

metal

of hemerythrin more complex

of iron-iron

this

of high

and nitrogen.

The MCD spectra are

AND BIOPHYSJCAL RESEARCH COMMUNICATIONS

and reduced

in a distorted Fe (II)

protein proteins between

tetrahedron,

states, is here

rubredoxin,

respectively

unusually

intense

examined,

380-390

1060

nm (Fig.

coordinated is

in

the high The MCD

(18,19). compared

and its

bands

3),

in marked

with

to those are

split

contrast

of with with

an

Vol. 51, No. 4,1973

BlOCHEMtCAL

the high spin iron in tranaferrin

AND BiOPHYSlCAL

RESEARCH COMMUNICATIONS

and conalbumin (Fig. 1).

The intense, bi-

phaaic signal of reduced rubredoxin at shorter wavelengths is not observed in the reduced forma of any of the other non-heme iron proteins examined. These distinctive

MCDsignals may prove characteristic

iron-sulfur

coordination.

more detailed

However, such conjectures remain to be explored in

investigations

now underway.

Systematic studies of synthetic properties

for high-spin tetrahedral

analoguea wilth structural

akin to those of the iron-sulfur

by Holm and his collaborators

proteins,

and electronic

such as reported recently

(20X and,of other complexes of iron with nitrogen

and oxygen liganda are needed to discern the precise basis of the spectra observed so far. REFERENCES 1. 2.

Ulmer, D. D. and Vallee, B. L., Biochemistry 2, 1335 (1963). Vallee, B. L. and Wacker, W. E. C., "The Proteins," H. Neurath, Ed., Vol. 5, Academic Press, New York (1970). 3. Ulmer, D. D. and Vallee, B. L., "Bioinorganic Chemistry," Adv. in Chem. Series 100, R. F. Gould, Ed., Am. Chem. Sot. Publ., p. 187 (1971). 4. Buckingham, A. D. and Stephens, P. J., Ann. Rev. Phya. Chem. l7, 399 (1966). 5. Schatz, P. N. and McCaffery, A. J., Quart. Rev. Chem. Sot. (London) 23, 552 (1969). 6. Kaden, T. A., Holmquiat, B. and Vallee, B. L., Biochem. Biophya. Rea. Consnun.46, 1654 (1972). 7. Ulmer, D. D., Federation Proc. m, 738 (1971). 8. Gray, H. B., "Bioinorganic Chemistry," Adv. in Chem. Series 100, R. F. Gould, Ed., Am. Chem. Sot. Publ., p. 365 (1971). 9. Bayer, E., Bather, A., Krauaa, P., Voelter, W., Barth, G., Bunnenberg, E. and Djeraaai, C., Eur. J. Biochem. 22, 580 (1971). 10. Ehrenberg, A. and Laurell, C. B., Acta Chem. gcand. 2, 68 (1955). 11. Feeney, R. E. and Komatsu, S. K., "Structure and Bonding," Vol. 1, p. 149, Springer-Verlag, Berlin (1966). 12. Aisen, P., Liebman, A. and Reich, 8. A., J. Biol. Chem. 241, 1666 (1966). 13. Gaber, B. P., Schllllnger, W. E., Koenig, S. H. and Aiaen, P., J. Biol. Chem. 245, 4251 (1970). 14. Price, E. M. and Gibson, J. F., J. Biol. Chem.247, 8031 (1972). 15. Poe, M., Phillips, W. D., Glickaon, J. D., McDonald, C. C. and San Pietro, A., Proc. Nat. Acad. Sci. U.S. 68, 68 (1971). 16. Poe, M., Phillips, W. D., McDonald, C. C. and Lovenberg, W., Proc. Nat. Acad. Sci. U.S. 65, 797 (1970). 17. Okamura, P., Klotz, I. M., Johnson, C. E., Winter, M. R. C. and Willisma, R. J. P., Blochemiatry 2, 1951 (1969). 18. Herriott, J. R., Sicker, L. C., Jensen, L. 8. and Lovenberg, W., J. Mol. Biol. 50, 391 (1970). 19. Phillips, W. D., Poe, M., Weiher, J. F., McDonald, C. C. and Lovenbarg, El., Nature 227, 574 (1970). 20. Her&wits, T., Averill, B. A., Helm, R. H., Ibira, J. A., Phillips, W.D. and Weiher, J. F., Proc. Nat. Acad. Sci. U.S. 69, 2437 (1972). 1061