Spectroscopic Methods in Bioinorganic Chemistry - American

Chapter 17

NMR of FeS Proteins Downloaded by UNIV MASSACHUSETTS AMHERST on September 26, 2012 | http://pubs.acs.org Publication Date: June 9, 1998 | doi: 10.1021/bk-1998-0692.ch017

1

2

1

Ivano Bertini , Claudio Luchinat , and Aileen Soriano 1

Department of Chemistry, University of Florence, via Gino Capponi 7, 50121 Florence, Italy Department of Soil Science and Plant Nutrition, University of Florence, P.le delle Cascine 28, 50144 Florence, Italy

2

The NMR linewidths of FeS polymetallic centers are discussed herein in terms of electronic relaxation rates and of the possibility of performing high resolution NMR spectroscopy on these systems. The linewidths depend on a single effective electron relaxation time, equal to or smaller than 5 x 10 s, which is our estimatefromthe iron-containing reduced rubredoxin. A summary of what is learned from NMR studies on the three polymetallic centers [Fe S ] , [Fe S ] and [Fe S ] , all having S'=1/2 ground state, is presented. -11

+

2

3+

2

4

+

4

3

4

The NMR of FeS proteins is largely the NMR of systems containing iron ions that are magnetically coupled in any of the naturally occurring polymetallic centers (Figure 1) (7,2). The synthetic models have permitted an enormous progress to be made in the understanding of these polymetallic centers (3,4). However, the effect of magnetic coupling on the electronic relaxation times and on the electronic structures of the synthetic systems has not been pursued as deeply as has been done on the corresponding proteins, one of the many reasons being the averaged symmetry of the synthetic models on the NMR time scale, at least at room temperature. The lack of any symmetry, the rigidframeprovided by the polypeptide chain, and the slow tumbling times resulted in great advantages for the direct investigation of iron-sulfur centers in the proteins themselves. The observability of the NMR lines in paramagnetic proteins depends on electron relaxation: the faster the electron relaxation, the sharper the NMR lines. In turn, electronic relaxation is efficient when there are excited electronic states close in energy. The ground state in tetrahedral F e is A j with S = 5/2. Therefore, there are no low-lying excited states. Estimates of zerofieldsplitting (ZFS) of the S = 5/2 multiplet in oxidized rubredoxin indicate that it is too small (1-2 cm" ) (5,6) to make effective Raman type (7) or Orbach type (8) electron relaxation. From the linewidths of ! H and H NMR signals we can estimate an effective electronic relaxation time at room temperature to be of the order of 10" - 5 χ 10" s. This makes NMR lines, for example of 3-CH2 protons of the coordinated cysteines, broad beyond detection. 3+

6

g

1

2

9

302

10

©1998 American Chemical Society In Spectroscopic Methods in Bioinorganic Chemistry; Solomon, E., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.

303 Indeed, the P-CH2 protons of the coordinated cysteines in oxidized rubredoxin have not been observed. The corresponding H signals, which should be a factor of 42 narrower, have been located at 800-1000 ppm downfield (9). The situation of F e is somewhat better: S = 2 makes the linewidths 2/3 those of S = 5/2. Furthermore, the ground state is E which is further split by low symmetry. Estimates of such splitting range between 600 and 1400 cm (10-12) which should provide relatively fast electron relaxation. Indeed, *H NMR signalsfromthe P-CH2 protons of the coordinated cysteines in reduced rubredoxin have been observed (9), but they are still very broad (in the 10 kHz range), and we can estimate an effective electronic relaxation time for F e to be around 5 χ 10' s. The effect of magnetic coupling on electronic relaxation times is very complex and only partly understood. In every polymetallic system with a magnetic coupling constant J larger than a few wavenumbers, we can probably define a single effective electronic relaxation time at each temperature (73). This should be equal to or smaller than that of the fastest relaxing ion by itself, in this case F e (Bertini, I.; Galas, O.; Luchinat, C.; Parigi, G.; Spina, G.; manuscript submitted, 1997). In the absence of new electronic relaxation mechanisms, only the mechanisms of the fastest relaxing metal ion should be effective for all metal ions due to magnetic exchange coupling. However, the new energy levels arisingfrommagnetic coupling may experience new and efficient relaxation pathways and thus decrease the overall electronic relaxation time. Therefore, whenever there is a F e ion, the effective electronic relaxation time can be set to be less than 5 χ 10" s. Thus, *H NMR can be attempted for any FeS system except [Fe2S2] , which contains only F e , and [Fe3S4]°, possibly because of the presence of chemical equilibria (Bertini, I.; Luchinat, C.; Mincione, G.; Soriano, Α.; manuscript submitted, 1997). Here we discuss three cases with resulting spin 5=1/2: [Fe S ] , [Fe S ] , and ^ S ^ " . The latter contains three F e ions. 2

2 +

5

Downloaded by UNIV MASSACHUSETTS AMHERST on September 26, 2012 | http://pubs.acs.org Publication Date: June 9, 1998 | doi: 10.1021/bk-1998-0692.ch017

-1

2+

11

2+

2+

11

2+

+

2

3+

3+

2

4

4

3+

4

+

The [Fe S ] case 2

2

J

A great advantage of H NMR spectroscopy is that we can follow the magnetic exchange coupling at room temperature whenever the ground state has total spin larger than zero. The Fe-S-Fe superexchange mechanism provides antiferromagnetic coupling (7). Thus, a system constituted by Sj = 5/2 (Fe +) and S2 = 2 (Fe ) will provide a ground state with «^=1/2 (the prime refers to spin states resulting from magnetic coupling). The larger spin, Sj = 5/2, will orient along the applied external magnetic field. The smaller spin S2 = 2 will, on the contrary, orient against the external magnetic field if J > kT because in the ground state the antiferromagnetic coupling prevails (Figure 2) (14,15). We recall the definition of and as the expectation values of Sj and S2 operators on the Sj and S2 spin wavefunctions, respectively. In the isolated systems, these expectation values are negative. In the coupled systems, Sj and S2 operate on the spin coupled wavefunction 5*. As a result of magnetic coupling, will be negative as for any isolated system and will be positive. Since the contact shift is proportional to - (through the positive proportionality constant which is the hyperfine coupling constant) (16), the contact shifts will be positive (downfield) for nuclei sensing Sj = 5/2 (Fe ) and negative (upfield) for nuclei interacting with S2 = 2 (Fe " ). Also, if the antiferromagnetic coupling constant J is much smaller than kT, all the $> levels are equally populated and there is no effectfrommagnetic coupling (73). When J = kT, intermediate cases are 3

Z

z

z

2+

Z

7z

3+

2

1-

In Spectroscopic Methods in Bioinorganic Chemistry; Solomon, E., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.


304

D

2 +

3 +

Figure 1. Cysteinate ligands coordinating F e / in rubredoxins (A); [Fe S ] (B) and [ F e S ] (C) in ferredoxins; and [ F e S ] (D) in ferredoxins (1+/2+) or HiPIPs (2+/3+), respectively. 1 + / 2 +

2

2

0/1+

3

1 + / 2 + / 3 +

4

4

4

Bo

S'=1/2

S2=2 Si=5/2

Figure 2. The 5^=1/2 spin resultingfromantiferromagnetic coupling between Sj = 5/2 and S2 = 2 spins orients along an external magneticfieldBQ. As a result, the #2=2 spin is oriented against the field, and its hyperfine interaction with nuclei changes sign.


305 +

1

observed. This is the case for [Fe2S2] clusters in ferredoxins (J = 200 cm" ). A representative NMR spectrum is shown in Figure 3. The less shifted cys P-CH2 signals (f - i) arise from the iron(II) site and their low temperature limiting shift is upfield, as expectedfromthe magnetic coupling scheme discussed above. The different linewidths between the iron(III) and the iron(II) sites are explained in terms of the different and values in the coupled system (75). The values are consistent with a single electron relaxation time for the pair, as expected. Our estimate, however, is about 2 χ 10" s which is shorter than that of iron(II) in rubredoxin. Apparently, there are additional relaxation mechanisms made available by the exchange coupling. Note that if electron derealization occurred, all signals would experience the same average value and all NMR signals would be downfield. In short, *H NMR provides a tool (/') for knowing whether or not there is a localized valence system with F e and F e and if so, as is the case for [Fe2S2] systems, (//) for recognizing the protons of each domain (77). Then, through the techniques of NMR of paramagnetic molecules we can (/) perform the sequence specific assignment of the signals of each domain and then (/'/) recognize which is the reducible iron (77). The reducible iron is that close to the surface of the protein on account of the large dielectric constant experienced by the region occupied by the solvent (18). In principle, it is possible to solve the whole structure in solution (19) as has been done for other Fe-S proteins (20-22).


12

3+

2+

3+

The [Fe4S4] case (oxidized HiPIP) J

The H NMR spectrum of oxidized HiPIP II from E. halophila shows four signals downfield and four upfield (Figure 4) (23). This has been reconciled with the idea of having Sj2 = 9/2 antiferromagnetically coupled with S34 = 4 where 7, 2, 3 and 4 denote each individual iron ion (24). The S12 9/2 occurs everytime there is a mixed valence (non localized) iron(II)-iron(III) pair (25). The S34 = 4 is due to the ferric pair. The values 9/2 and 4 for the S12 and S34 spin pairs are the result of applying the Heisenberg Hamiltonian, % = JSj · Sj , to each pair of / and j metal ions. Under antiferromagnetic coupling (J> 0), if Jj 2 is the smallest, Sj and S2 are forced to be ferromagnetically coupled with Sj2 = 9/2. This is because not all spins can be antiferromagnetically coupled one to the other in a close circular arrangement. Therefore, the pair experiencing the smallest antiferromagnetic coupling indeed acquires a ground state typical of ferromagnetic systems. S34 has a value smaller than 5, and indeed is 4, for a very large range of J values. Actually, to explain the low temperature hyperfine values with F e and the EPR data, it has been proposed that the ground state is the following linear combination of |9/2,4> and |7/2,4> wavefunctions (26,27): =

57

Ψ = 0.95|9/2,4> + 0.31|4,7/2>

(I)

This ground state is obtained with a J coupling scheme with C symmetry. Thus, the two cysteines bound to the mixed valence pair will experience downfield shift and the two cysteines bound to the ferric pair will experience upfield shift. The sequence specific assignment of the cysteines recently became available (23) and shortly after, the solution structures of this type of proteins were solved (20-22) (Figure 5). s


306


a-d Ηβ Cys 111,1V

f-i Ηβ Cys 1,11

+

Figure 3. *H NMR spectrum of the [Fe2S2] -containing reduced ferredoxin from spinach (43). The eight signals (a-d; f-i) arisefromthe β-ΟΗ2 protons of the four cluster-coordinated cysteines. The four far downfield signals (a-d) originate from the ferric site whose S = 5/2 spin is oriented along the magneticfield;the less shifted signals (f-i) originate from the ferrous site whose ground 5 = 2 spin is oriented against thefieldin the ground state (see Figure 2). The low temperature limiting shift value of the latter signals is in fact upfield. The sequential assignment of the signals to the cluster-bound cysteines is also shown.

W Ηβ2 Cys IV Χ Ηβ1 Cys IV Υ Ηβ2 Cys I Ζ Ηβ1 Cys I

Α Ηβ2 Cys III Β Ηβ2 Cys II Ch^1 Cys III D Ηβ1 Cys II

πι

lui

II 100

80

60

40

δ l

-20

20

-40

(ppm) 3+

Figure 4. U NMR spectrum of the [Fe4S4] -containing oxidized HiPIP II from E. halophila (23). The eight signals arise from the P-CH2 protons of the four cluster-coordinated cysteines. The four downfield signals (a-d) originate from the mixed-valence F e - - F e - pair whose ground state Sj2 9/2 spin is oriented along the magneticfield;the four upfield signals (w-z) originatefromthe ferric F e - F e pair whose ground state S'34 = 4 spin is oriented against the field. The sequential assignment of the signals to the cluster-bound cysteines is also shown. 2

3+

5+

2

5+

=

3+


307

Some oxidized HiPIPs may have a more complicated behavior owing to the possibility that electron derealization occurs in more than one pair of iron ions. This has been reviewed in a previous ACS series book (28). The ! H NMR lines in [Fe4S4] systems are very sharp, and particularly much sharper than in [Fe2S2] systems. Again, the data are consistent with a single τ value, much shorter than that of iron(II) in rubredoxin. 3+

+

8

+


The [Fe3S4] case This case is interesting because the cluster contains only iron(III) ions, as in the oxidized [Fe2S2] ferredoxins, the latter giving very broad NMR lines. The ground state of the [Fe S4] cluster has 5*=1/2 with g values around 2.02, 2.00 and 1.97 (1,29). Its *H NMR lines are almost as sharp as in [Fe4S4] systems (compare Figures 4 and 6). Apparently, a dramatic shortening of the overall τ with respect to the isolated F e ion by exchange coupling has occurred and the shortening is sizable also with respect to the [Fe S2] case. This may be a general phenomenon due to the existence of excited states with the same spin as the ground state in trinuclear (and higher nuclearity) clusters. It is possible that the sharpening of the NMR signals increase with the nuclearity of the cluster. The NMR investigation of [Fe3S4] systems has been recently extended to a number of proteins, and the ambiguities in the assignment resolved (30-36). It appears that the [Fe3S4] environment in 3Fe4S proteins is slightly differentfromthat of [Fe3S4] in 7Fe8S proteins (Figure 6 A and B, respectively). This has been related (36) to the difference in primary sequences (Figure 7) (37). The 3Fe4S proteins look more symmetric, as the protons of one cysteine are far downfield shifted and those of the other two cysteines are less shifted (Figure 6A). In the [Fe3S4] cluster of 7Fe8S proteins there is also one far shifted cysteine p-CH2 pair, but the other two P-CH2 pairs are more inequivalent as one proton of a pair is slightly upfield (Figure 6B). We have proposed (36) that, with one possible exception (31), Cys IV in the primary sequence is the one far shifted downfield and Cys I is shifted around zero or upfield (Table I). We have also drawn energy diagrams for the two cases (Figure 8) (36) depending on the J values in the following Hamiltonian: 2+

+

3

3+

8

3+

2+

2

+

+

+

+

κ=Ji2$r h+Ji£i'S3+J23S2S3

() 2

The experimental temperature dependence is also consistent with the theoretical expectations from the proposed electronic structure, both for the more symmetric (32,33) as well as for the less symmetric clusters (35,36). In Figure 9, the experimental and calculated values for the less symmetric cases are reported. In the more symmetric cases, the shifts for the B, / , 7, WXtnà to be similar to their average value. Concluding remarks ! H NMR spectroscopy is a powerful tool for monitoring the contribution of each metal ion to each spin level in a magnetic coupled system. This permits the investigation of the valence distribution in Fe^m polymetallic centers, where average oxidation states of 2+, 2.5+ and 3+ have been found. If the sequence specific assignment of the protein is available, this thenframesthe valence distribution inside the protein structure. At this point, on one side we can proceed to obtain the solution structure of the protein,



308

Figure 5. Schematic representation of the solution structure of the [Fe S4p" "containing oxidized HiPD? IfromE. halophila (27). The thickness of the tube is proportional to the backbone RMSD. Note the high resolution even in the proximity of the paramagnetic center. +

4

1 Cys I

1

δ(ρρηα)

1

1

1

1

25

20

15

10

1

5

Γ

0

Β

i

1

δ(ρρηΐ)

2

5

1 2

0

1

!

15

10

1

5

Γ 0

Figure 6. NMR spectra of the [Fe3S4] -containing oxidized ferredoxins from D. gigas (3Fe4S ferredoxin) (A) (33) and R. palustris (7Fe8S ferredoxin) (B) (36). Signals in A and Β are labelled according to the sequence-specific assignment in B. The sequential numbering of the corresponding cysteines is also shown. The sequential numbering of the [Fe4S4] cluster in Β is omitted for clarity. Cys IV βCIÏ2 protons are most downfield and show a Curie-type temperature dependence in both cases. Cys I and III P-CH2 protons are almost equivalent and all antiCurie in A, whereas they are strongly inequivalent in B, with signal I being upfield with a pseudoCurie behavior (see also Figure 8). +

2+



309

Figure 7. Different primary sequences characterizing most 3Fe4S (A) and 7Fe8S (B) ferredoxins (36,37). Arabic numerals refer to the type of cluster, and roman numerals refer to the sequential cysteine ordering in the cluster.

Table I. Summary of the available NMR data on 3Fe4S and 7Fe8S ferredoxins. Cys-l3 antiCurie

Cys-IIl3 antiCurie

Cys-IV3 Curie

-18, -3.5

-15.2,-8.5

29.2, 24.5

T. litoralis (32,39)

17.4,4.9

16.6, 8.1

29.2,24.0

P. furiosus b (31,32)

19.7,17.1 Curie

14.3, 5.2

23.6,11.8 antiCurie

b

D. gigas (33)

b

(0-10), (0-10) 21.8, (0-10)

c

P. putida (30,40)

31.8, 26.3

4

Cys-IV antiCurie

e

9.8, 9.3rf-

4

4

Cys-III antiCurie

4

Cys-I antiCurie

Cys-II antiCurie

e

9.8,9.3rf>

(0-10), (0-10) (0-10), (0-10)

17.3, (0-10) rf 15.3,(0-10) 16.9, 9.1

15.2,5.6

d

A. vinelandiif (30)

(0-10), (0-10)

21.1,8.6

B. schlcgclii S (35)

(0-9), (0-9)

18.3,6.6

32.2, 23.4

10.35,-9

11.1,9.0

15.8,9.7

15.9,5.2

Κ 5.8

25.8, 9.5

29.6,24.4

10.2, 9.6

10.6,9.7

17.15,9.3

15.9, 6.0

17.1,7.5

21.7,18.9

14.2, 7.9

10.9,9.3

17.9,9.2

14.0, 6.3

d

R. palustris >S (36) D. ambivalens & (41) P. ovalisu* (42)

-0.8

20.0, 9.2

(0-10), (0-10) 22.3, (0-10)

M. smegmatis & (0-10), (0-10) 22.9, (0-10) (42) T. thermophilus d>i (0-10), (0-10) 21.8, (0-10) (42)

32.2, 26.1

30.9,25.7

e

10.5 , (0-10) 10.5 e

e

a

(0-10) 17.7, (0-10)7 15.7, (0-10)7 16.2, (0-10)7

e

29.8,21.5

11.0 , (0-10) 11.0 , (0-10) 17.1,(0-10)7

29.0,21.1

11.1 , (0-10) 11.1 (0-10) 17.4,(0-10)7 17.0, (0-10)7

a

e

e

c

Chemical shifts in ppm and their temperature dependence; * 303 K; 281 Κ;rfsequence specific assignment proposed in (36) ; only one signal or one pair is observed. It is impossible to discriminate between Cy s-IV andCys-I ;/280K;^ 298 K ; pseudoCurie; '300 K;7 the assignment to Cys-II or Cys-III could be reversed. e

4

h

4

4


4

310

-5700


-3750

|3,5/2> ^ ^ i .

J l 2 = 285 cm-1 J l 3 = 300 c m "

-3900

J l 3 = J23 = 300 cm-1

1

-3950

_l

-4000 320

315

310

300

305

285

1

J23 (cm" )

290

L_

295

300

Jl2 (cm-1)

D

300

305

310

315

320

300

J l 3 = 300 c m "

1

«Ï23 = 320 c m "

1

295

290

285

J l 2 (cm- )

J23 (cm-1)

1

Figure 8. Calculated eigenvalues for the two lowest lying energy states of the Hamiltonian (equation 2) in [Fe3S4] clusters for different J values. Β and C refer to the more symmetric case encountered in 3Fe4S ferredoxins (two equal J values), while A and D refer to the more asymmetric case encountered in the [Fe3S4] cluster of 7Fe8S ferredoxins (three different J values). Cases A and Β can be described by Sj2 (or S23) =3 (or 2) and S3 (or Sj) = 5/2, respectively, while in the asymmetric C and D cases these are no longer good quantum numbers. The set of J values that qualitatively reproduces the experimental temperature dependence for the asymmetric cases (see Figure 9) is shown both at the extreme left of A and at the extremerightof D, which are in fact coincident. +

+



311

J 1

0.0

3.2

3.3

3.4

-ι ι— 2.0 4.0 1/TX103 (1/K)

3.5

1 / T x l 0 3 (1/K)

Figure 9. Experimental (A) and calculated (B) temperature dependences for the hyperfine shifts of the cysteine p-CH2 protons in asymmetric [Fe3S4] clusters (35, 36). The curves in Β are calculated using Jj2 = 285 cm , J] 3 = 300 cm" and J23 = 320 cm" in the Hamiltonian (equation 2). The signal labelling refers to Figure 6B. +

-1

1


1

312 and on the other, the factors determining a given valence distribution and overall reduction potential can be investigated (38).


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Spectroscopic Methods in Bioinorganic Chemistry - American

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