A new cluster model for the FeMo-cofactor of nitrogenase

Vol. 88, No. 4, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

June 27, 1979

Pages 1454-1461

A NEW CLUSTER MODEL FOR THE .FeMo-COFACTOR OF NITROGENASE Boon-Keng

Tea+ and' Bruce

A. AverillS

+Bell Laboratories, Murray'Hill, New Jersey 07974 and IDepartment of Chemistry, Michigan State University, East Lansing, Michigan 48824

Received

May 25,

1979

SUMMARY A new structural model for the FeMo-cofactor of nitrogenase, consisting of two Fe4S4 'clusters bridged by an S2M02 unit, is proposed. Available chemical, spectroscopic, and EXAFS data are shown to be consistent with the proposed structure. In particular, EXAFS data are in agreement with m(Mo-Fe):n(Mo-S) of either 2:4 or 3:4; comparison with known MO-Fe-S and Fe-S systems leads us to favor the former. Based on the proposed structural model, a possible mechanism of reduction of N2 is suggested. INTRODUCTION Recently protein the

a low molecular

of nitrogenase

FeMo-cofactor,

sulfur

approximately

S = 3/2

unit.

reported

absorption to probe the

model

the

MoFe-protein compound

of k and the site

3.8 model

(5),

S at 2.35 the

(3),

the

structural

that

An examination

of the

0006-291X/79/121454-08$01.00/0 Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in anyform reserved.

1454

X-ray

of EXAFS

has been demonstrated

A comparison

MO-Fe-S

of the

EXAFS

as indicating

that

the MO

namely

the

MO of the

features,

consistent

spin-coupled

and a polynuclear

i as nearest is

labile

have indicated

ability

an Fe-S cage (cubane-like)

Fe at 2.72

FeMo-cofactor

(5)

species,

and 6 acid

EXAFS (extended

(4),

the MoFe-

to the MO have yet

of the molybdenum

(I).

from This

a novel

The unique

FeMo-cofactor

into

iron

into

except

was interpreted

certain

(2).

(2)

assignable

[Mo2Fe6S9(SC2H5)81J-,

i and 3.0

been proposed.

incorporated

method

environment

was incorporated

for

studies

spectroscopy.

FeMo-cofactor

in each shared

cofactor

Spectroscopic

unambiguously

structure)

immediate

characterized 8 non-heme

by any spectroscopic fine

has been isolated

to contain

6 Fe per MO are No features

been detected

yet

is

cofactor

and partially

atoms per molybdenum.

that

for

(1)

weight

neighbors. with

available

the

that

structure

with

No structural available

EXAFS and other

data

has

spectroscopic

Vol. 88, No. 4, 1979

data

has convinced

and leads for

BIOCHEMICAL

us that

directly

the

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

an alternative

to the postulation

interpretation

is equally

of a chemically

reasonable

plausible, structure

FeMo-cofactor.

RESULTS AND DISCUSSION We propose cubes via with

four

the

for

that

While

p-sulfide

EXAFS data

reported with

a structural

the

to Mo is

as well

observed

for

identity

of the

known,

of the cofactor

solvent

or additional acid

classes

observed

S = 3/2

center

quadrupole and the

unit

(11,12). doublets

FeMo-cofactor

(9)

for

of M

(12,13).

unit

in the

for thus

We assume that

1455

bridged

by symmetry

of @, MoS4=,

into

of the 6 Fe6 -via

to give

the spin

of

to produce

conceivably

spectra

(7).

upon

hydrolysis

result

6 Fe associated

assumed

Mossbauer

and 6 f 2 S=/Mo

from Clostridium

coupling

the

consistent

as is coordination

divided

could

is

to the 6 FeB not

the MoFe-protein

accounting

The 2 FeA are

MoFe,$,2

be expected

are clearly

is consistent

and the stoichiometry

Further,

would

two Fe4S4

by N-methylformamide

(a),

Antiferromagnetic

FeAS2MoS2FeA

observed

ligands

possible

conditions

model

8 f. 1 Fe/MO,

to the molybdenum.

atoms

(lo),

data

coordinated

of protein

by Zumft

diamagnetic

(1):

bridges

This

stoichiometry

ligands

certainly

of 6 FeB and 2 FeA.

a presumably

3/2

is

The iron

the

FeMo-cofactor terminal

in JJ,.

spectroscopic

Thus,

oxidizing

reported

pasteurianum.

the

the molybdenum

as shown

as other

groups

under

in which

(6),

substitution

removal

as recently

units

the FeMo-cofactor.

not

by dilute

model

rise

with

in the the S =

to the simple

of the MoFe-protein density

on the FeB

(11)

Vol. 88, No. 4, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

k (i-'1

I

4

I

6

A

10

8

I

12

k (%-'I Figure

1.

Fourier-filtered (solid curves) EXAFS data (5,15) and the leastsquares fits (dashed curves) with theoretical amplitude and phase functions (16,17) for the MO k edge of (a) nitrogenase and lb) [M02Fe6Sg(SC2H5)813-.

1456

Vol.

88,

No.

BIOCHEMICAL

4, 1979

atoms resides

in orbitals

contributions

from the

A direct

"-' A

for

Fourier

transforms

the

k3X(k)

distances. did

dashed

not

curves

be 2.35

i,

These

functions

three-term squares

fits

for

does

not agree

reported

nitrogenase,

while

We believe has fewer

that

Knowing assuming we estimate

that

way of estimating

iron

is

clear

nearest

neighbors

We proceeded

m MO-Fe and n MO-S

at significantly

The best

nitrogenase,

fits

longer

are shown as

are determined

and 2.32

crystallographic

The

can be assigned

to L.

involving

and 2.74

results

upon parameterized

at least

earlier

that the

there

the sulfur that

the

(based

and 0.82

neighbors

that

with

for

of 0.79 latter

there

are

Plots

the number

not

to i for

l and the

with

3.

reported that

atom in nitrogenase than

three

(and the

1457

neighbors

minima

at m/n =

The former

value

earlier

for

(5)

for

l.

FeMo-cofactor)

similar

in ,J, and in nitrogenase,

in nitrogenase. is

the

A.

EXAFS are

2.4 MO-Fe interactions

and

(sum of

MO-Fe interactions

to the

of nearest

than,

reported

in the MoFe3 model,

certainly

better

give

i as shown in Figure

identical

amplitude

of the chi-squares

of m(Mo-Fe):n(Mo-S)

contributions are

if

(m/n = 3.01/3.79)

is

(18)

theoretical

as good as,

(5).

for

the molybdenum

iron

for

as a function

nitrogenase with

appreciably.

at k = 10

It

compared

wave due to MO-S'

differences

distance

fewer

the

la and lb).

to the MO-Fe distances.

model

are

of residuals)

0.66

The shorter

a two-distance

for

(5).

(16,17)

fits

orbital

occur

Figures

The MO-S and MO-Fe distances

agreement

two-term

which

(6.

atoms),

respectively,

EXAFS study

to acquire

substantial

nodes

sulfur

the fit 1.

2.

are

has significantly

of a third

in Figure

in substantial

phase

with

improve

and 2.71

previous

longer

of nearest

data

beat

and X, respectively

and the

Inclusion

distance

COMMUNICATIONS

EXAFS data

there

the

shown in Figure

number

filtered

that

especially

of nitrogenase

to the

to fit

I,

are

MO-S distances

(relative

RESEARCH

by symmetry

Fourier

and t shows

nitrogenase

the MO site

forbidden

of the

envelopes,

and 8.4

that

(15)

of nitrogenase

in the amplitude

are

BIOPHYSICAL

Fe* or MO atoms.

comparison

MoFe protein

to the

which

AND

to assume that

An alternative the

Debye-

Vol. 88, No. 4, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

0a

I, 8 F-J

,

,

,

I, 8 RI

I

(,J)

I

‘$’

1458

I

I,,,, 8

lo 0

Vol. 88, No. 4, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNlCATlONS

60

-25

N I TROGENASE

I

I

30

0.25

I

0.5 m (MO-FeVn

Figure

3.

Waller

factors

ratio

MO-Fe peaks

for Fourier

transform

should

give

gives

however,

can be obtained

parameter--the

for it

iron

magnitudes relative

(19)

(cf. -

number

factor--from to nitrogenase.

In short,

given

the present

certainly

or 3:b.

Comparison

the precise

consistent of the

with

1459

Using

The best

The the

l as a estimate,

of a single

together

and highwith

the

MO-Fe interactions

EXAFS data on nitrogenase, of MO-Fe and MO-S bonds.

m(Mo-Fe):n(Mo-S)

EXAFS of the model

2a and 2b) for

atoms.

MO-S and 2.3(4)

number

cases.

ferredoxins

This,

3.5(5)

are

of iron

bacterial

gives

The data

Figures

the transferability

of m/n = 0.66,

to determine

in both

in nitrogenase.

by assuming scale

proteins

nitrogenase. difficult

the

are similar

1.9 MO-Fe interactions

overall

value

is

(MO-S)

the MO-Fe interactions

standard

fitted

10

Plot of c2 (sum of squares of residuals of least-squares fits) vs. m/n where m and n are the numbers of MO-Fe and MO-S bonds, respectively for nitrogenase (solid curve) and [Mo2Fe6Sg(SC2H5)813(dashed curve).

of the

potential

I 1.0

0.75

compound

of either l with

2:4

nitrogenase

Vol. 88, No. 4, 1979

leads

BIOCHEMICAL

us to believe

was reported

that

earlier

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the former

is more probable

of the

proposed

structural

model

mechanism

of nitrogen

binding

MO (e.g.,

Mo(IV),

and the two FeA should

in which result

would

would

reasonable

by a tri-iron

cluster

reactions

increases and related

the

intermediate

clusters

involvement

are

are

chemical

and

process

of ammonia.

unit,

to

N-NH2 has

fixation

moles

and

rise

MoBr(N*H)(ddpe)*, the

attractiveness

give

intermediates

by the catalytic

in the

The net

of the N-N bond.

These

by a trimetallic

Fe4S4 units

in a complex

(20).

atom would

wouldyieldtwo

The direct

linear

and result

in the nitrogen

suggested

(23).

ubiquitous

this

In fact,

fixation is

configuration

the two Fe4S4 cubes

in Mo(N*)*(ddpe)*,

and protonation

is chemically

synthesize

via

nitrogen

to be important

of nitrogen

in a folded

bonding,

manner

respectively.

the mechanism

transfer

(21)

in a possible between

and activation

of the terminal

been reported

apparently

weakening

observed

reduction

which

interactions

to the two FeA atoms

in a stepwise

[MoBr(N2H2)(ddpe)2]+,

Further

result

MO-N=N + MO-N=NH + MO-N-NH*.

to those

G results

Bonding

FeA-Mo-FeA

is a-bonded

protonation

intermediates

latter,

N2 to the MO in the expected

the

be a significant

successive

recently

Binding

perturb

of electrons

similar

and reduction.

unit.

the dinitrogen

Injection

the

d*)

the FeAS2MoS2FeA

MO-N=N fashion

the

(5).

Consideration

for

than

That

as proposed

reduction

here,

of RC=N

of the well-known binding

(22).

and

and electron

of the model.

Attempts

to

in progress.

ACKNOWLEDGEMENTS Support National Grants

of this

Science Office

research

Foundation

(5901-0410-8-0175-0)

by Bell

Laboratories

(CHE-7715990) (B.A.A.)

(B.K.T.),

and the

and the USDA/SEA Competitive is

gratefully

acknowledged.

REFERENCES AND NOTES 1. 2.

Shah, V. K., Brill, W. J. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 3249-3253. Rawlings, J., Shah, V. K., Chisnell, J. R., Brill, W. J., Zimmermann, Mijnck, E., Orme-Johnson, W. H. (1978) J. Biol. Chem. 253, 1001-1004.

1460

R.,

Vol. 88, No. 4, 1979

3.

Cramer,

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

S. P.,

Hodgson, K. O., Gillum, W. O., Mortenson, L. E. (1978) 100, 3398-3407. Cramer, S. P., Gillum, W. O., Hodgson, K. O., Mortenson, L. E., Stiefel, E. I., Chisnell, J. R., Brill, W. J., Shah, V. K. (1978) J. Am. Chem. Sot. 100, 3814-3819. Wolff, T. E., Berg, J. M., Warrick, C., Hodgson, K. O., Holm, R. H. (1978) J. Am. Chem. Sot. 100, 4630-4632. The possibility of bridging p-SR units cannot be ruled out, although the identity of R in the cofactor is not clear. Complexes containing FeS MoS units have been prepared; these will be described elsewhere. Aci 3 -la 6 ile sulfide assays of Fe-S proteins are often low; further, we have found that MO-S units do not give stoichiometric results under the normal assay conditions (S. R. Tonsager and B. A. Averill, submitted for publication). A minor variation of II in which the 2 S= opposite the FeS2Mo units are absent is also possible. This is especially likely if the protein uses ligating atoms more electronegative than sulfur. This would increase the lability as well as raising the redox potential. See Johnson, R. W., Holm, R. H. J. Am. Chem. Sot. (1978) 100, 5338-5344. Zumft, W. J. (1978) Eur. J. Biochem. 91, 345-350. For example, coupling of 3 high spin Fe(II1) and 3 high spin Fe(I1) could yield a net S = 3/2. Miinck, E., Rhodes, H., Orme-Johnson, W. H., Davis, L. C., Brill, W. J., Shah, V. K. (1975) Biochim. Biophys. Acta 400, 32-53. Huynh, B. H., Miinck, E., Orme-Johnson, W. H. (1979) Biochim. Biophys. Acta 527, 192-203. ItBis not immediately obvious how antiferromagnetic coupling of the 6 tectable hyperfine field at the bridging FeA :;t,;~"' ;o;$,~~"e~~cen~ i% MO hyperfine is detecta e in the EPR spectrum of MoFe-protein obtiined from organisms grown on $4 MO, arguing that the S = 3/2 species does not interact appreciably with the MO sites (14). Further, quantitation of the Mijssbauer data (12) suggests that the quadrupole doublet species is due to only one Fe per S = 3/2 unit. (a) Orme-Johnson, W. J., Jacob, G. S., Henzl, M. T., Averill, B. A. (1977) Bioinorganic Chemistry-II, Raymond, E., ed., ACS Adv. Chem. Ser. 162, 389-401; (b) Palmer, G.,Multani, J. S., Cretney, W. C., Zumft, W. G., Mortenson, L. E. (1972) Arch. Biochem. Biophys. 153, 325-332. The da$a were taken from Reference 5 except that the usual k (rather than k ) weighting was employed. (a) Teo, B. K., Lee, P. A., Simons, A. L., Eisenberger, P., Kincaid, B. M. (1977) J. Am. Chem. Sot. 99, 3854-3856; (b) Lee, P. A., Teo, B. K., Simons, L. (1977) J. Am. Chem. Sot. 99, 3856-3859. Teo, B. K., Lee, P. A. (1979) J. Am. Chem. Sot. 101, 0000. In particular, a model in which all 6 Fe of the S = 3/2 unit interact equally with the MO at normal MO-Fe distances (e.g., a corner-sharing dicubane structure involving an Fe3MoFe3 cluster) seems to be totally at variance with the EXAFS data. Teo, B. K., Shulman, R. G., Brown, G. S., Meixner, A. E., J. Am. Chem. Sot. submitted for publication. A somewhat different but relevant mode of (u + r) metal-dinitrogen binding has been observed in (n5-C5H5) (n1,n5-C5H ) Ti2-N2-(n5-C5H5)2 (n5,n5-ClOH8) Ti2whereN2 is u-bonded i! o one Ti an I! r-bonded to two different Ti atoms. (G. Pez, private communication). Another relevant example is Fe3(RCzN)(C0)9 where the RC-N is u-bonded to one iron atom and n-bonded to the remaining two iron atoms (23). Chatt, J., Pearman, A. J., Richards, R. L. (1975) J. Organometal. Chem. 101, c45-c47. Thorneley, R. N. F., Eady, R. R., Lowe, D. J. (1978) Nature (London) 272, 557-558. Andrews, M. A., Kaesz, H. D. (1977) J. Am. Chem. Sot. 99, 6763-6765.

J. Am. Chem. Sot. 4. 5. 6. 7.

8.

9. 10. 11. 12. 13.

14.

15. 16. 17. 18.

19. 20.

21. 22. 23.

1461