J. Phys. Chem. 1993,97, 3017-3021
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Conformational Distribution in Protein-Bound [3Fe-4S]+ Clusters: CW and Pulsed EPR and 57Fe ENDOR of D. gigas Hydrogenase Chaoliang Fan, Andrew L. P. Houseman, Peter Doan, and Brian M. Hoffman’ Department of Chemistry, Northwestern University, Evanston, Illinois 60208 Received: October 29, 1992; In Final Form: January 25, 1993
This paper discusses 9- and 35-GHz EPR spectra of the Desulfovibriogigas [3Fe-4S]+ cluster and also describes 35-GHz C W 57FeENDOR measurements of isotopically enriched protein. The EPR spectra of [3Fe-4S]+ clusters typically cannot be described by a well-defined g tensor. Their properties instead can be modeled with a formal treatment that assumes there is a distribution in the gvalues. This treatment implicitly assumes that the EPR spectra of such clusters are controlled by a distribution in protein conformation and cluster structure, but the validity of this assumption has never been confirmed by an experiment that correlates the EPR spectrum of a cluster with a microscopic cluster property. We now report such a correlation. We find that the EPR and the 57FeENDOR spectra for the D. gigas cluster can be described jointly in terms of a superposition of contributions from a bimodal distribution in cluster forms. Decomposition of the EPR spectra indicates that the major form has a well-defined structure as reflected in a well-defined g tensor with principal values 2.032, 2.024, and 2.016. The other, minority form shows a significant distribution in g tensor values. 57FeENDOR measurements show that the iron ion of the cluster with the largest hyperfine coupling has quite different properties in the two forms, thus confirming the implicit assumption in decompositions of the EPR spectra that the protein exhibits different substates in which the cluster exhibits differing structures and properties.
Inboduction
Materials and Methods
The structure of [3Fe4S]+ clusters has been determined,’ and the main feature of the spin coupling among the three ferric ions of the oxidized l l + ) state of the cluster have been
The as-purified D. gigas hydrogenase samples and those with 57Feenrichment were prepared as previously de~cribed.~ X-band (9 GHz) pulsed EPR spectra were obtained on a locally built spectrometer? CW EPR and ENDOR spectra at 35 GHz were recorded on a spectrometer described elsewhere.IO EPR simulations were performed with the program QPOW.” The ENDOR transition frequencies12for S7Fe( I = l/2) are given to first order by the equation
I4 RS’
S ‘’
established.2 Nonetheless, fundamental questions about the properties of this state remain. In particular, the EPR spectra of [3Fe4S]+ clusters in proteins are anomalous in that they typically cannot be described by a well-defined g tensor,’-$ but rather appear to reflect a distribution in tensor values. Such spectra have been modeled with a formal treatment based on a distribution in g values that in turn can be described as arising from a distribution in the relative values of the spin-exchange coupling between iron ions and the single-ion zero-field ~plittings.~ This treatment implicitly assumes that the protein adopts a range of conformationalsubstates6in which the cluster exhibits slightly differing structures with different properties. However, to date such a treatment has not been validated by any experiment that correlates the EPR spectrum of a cluster form with a microscopic property of the cluster in that form. To address this issue, we have performed ajoint EPR and 57FeENDOR study of the [3Fe4S]+ cluster of Desulfovibrio gigas h y d r o g e n a ~ e . 4 * ~ ~ ~ ~ ~ We find that the 9-GHz pulsed and 35-GHz CW EPR spectra for the D. gigas [3Fe-4S]+ center can be described in terms of a bimodal distribution in cluster forms. The major form has a well-defined structure as evidenced by a well-defined g tensor. The other, minority form apparently is associated with a more plastic conformation, resulting in a significant distribution in g tensor values. The link between the formal decomposition of the EPR spectrum and microscopic properties of the clusters is provided by 35-GHz CW $’Fe ENDOR studies, which show that the cluster iron ion with the largest hyperfine coupling, denoted Fe( 1). has quite different properties in the two forms. OO22-3654/93/2097-30 17$04.00/0
where AFCis the orientation-dependenthyperfinecouplingconstant and is the nuclear Larmor frequency. Usually Y F ~C AFc/2 and the ENDOR spectrum is a Larmor-split doublet, centered at AFe/2 and split by 2VFe (1.7 MHz at Bo = 12 500 G). ENDOR simulations were performed12as described. This study used two different samples with slightly different bulk physical properties that result in different resonant microwave frequencies. Thus, any feature in the EPR spectrum will occur at a different field in the two samples, and we must compare their EPR and ENDOR spectra by referring to g value rather than field. Because the g anisotropy is small, we must specify field position in gvalue to 4 decimal places in order to distinguish field positions adequately. In this paper, we report g2 = 2.024; thus, we definegz 2.0240 and report all observed gvalues with respect to g2. We emphasize that the g values are not so accurate to warrant 5 significant figures. However, the precision of the field measurementsallows us to take 6g = gob- g2 to this many decimal places. This use of precise g values also facilitates comparison of X- and Q-band EPR. Results and Discussion
EPR. The hydrogenase from Desulfovibrio (D.) gigas as isolated contains one nickel center, one paramagnetic [3FAS]+ cluster, and two EPR-silent [4Fe4S]+ ~ l u s t e r s ,The ~ ~ ~nickel Q 1993 American Chemical Society
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The Journal of Physical Chemistry, Vol. 97, No. 12, 1993 I
I
Fan et al.
TABLE I: g TenmmP rad We HyperAae Tensod for the [3Fe-4Sr Clmter of D. OQUH Form I (70%) p(l) = (2.032,2.024,2.016], A l a 30 MHz
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AFe(l,I)= [51.4 (S), 41.2 (2), 41.2 (2)]; (a 30°, 4 = 0 (lO)O, y = 0)C AFe(l,l)= [SO, 44,381 (M&ssbauer)d A"(2,l) % 18-22 MHz A"(3,l) 3 MHz ( M W b a u ~ r ) ~ Form 2 (30%) p(2) = [2.029,2.017,2.003], AW= [39, 51,761 MHz A"( 1,2) 39 MHz AFe(2,2)= 23 MHz
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-
(Am,
O g values, line widths and percentage contributions to the simulations of EPR spectra were obtained as described in text. "Fe hyperfine tensors labeled as AFe(i&,i = 1-3 is the Fe site,j = 1,2 is the cluster form. For definition of Euler angles, sa ref 12. See ref 7.
I
I
2.04
2.02
2.00
g-value F i i 1. Experimental and simulated EPR spectra. (A) X-band threcpulse echo-detected EPR. Experimental conditions: z/2 pulse, 80 ns; microwave frequency 9.4823 GHz; 7 value, 0.280 p; Tvalue, 23.28 ja;
20samples/point, 10.2Hzrepetitionrate,512points(1000s). (B)Solid line: Q-band CW EPR spectrum. Experimental conditions: microwave 1.6 G. Dashed lines: frequency, 35.4 GHz; field modulation decomposition of simulated absorption envelope of form I and form 2 clusters. Simulation parameters are described in the text. (C) First derivativeof (e). (D) Solidline: simulatedfit-derivativeEPRspcctrum. Dashed lines: decompositionof simulated first-derivativeEPR s p e d " intocontributionsfromform 1 and form 2 clusters. Simulationparameters are described in the text.
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center exhibits an EPR signal, termed Ni signal A, with g = [2.31, 2.26, 2.021. The EPR absorption envelope of the [3Fe4S]+ cluster as obtained with Q-band CW-EPR (Figure 1A) and X-band two-pulse echo-detected EPR (Figure 1B)shows a sharp signal at g = 2.02 with well-defined shoulders on both sides, as well as a tail extending to high field. Although the Ni A signal extends to g,i, = 2.02, its amplitude is much weaker than that of the [3F&S]+ cluster signal, so that no features in Figure 1 are associated with it. Figure 1C presents the derivative of the 35-GHz spectrum, which emphasizes the highly resolved values of the sharp spectrum. As the X-band and 35-GHz spectra are essentially indistinguishable when plotted as EPR intensity vs g value, all the features in the EPR spectra must be caused by anisotropy in the g tensor rather than by hyperfine interactions or spin-spin interactibns with the paramagnetic Ni A site in the protein. The sharp signal from [3Fe-4S]+ clusters, to be designated form 1, can be simulated with the well-defined rhombic g tensor, g = [2.032,2.024,2.0161, and an isotropic line width, AW 5 30 MHz. However, such a simulation does not reproduce the highfield tail in the spectrum. This tail is not associated with partially degraded protein, for spectra from different protein preparations are superposable and in any case such a tail is normal for the [3F&]+~luster.~~Instead, thehigh-field tailintheEPRspectra of Figure 1 appears to be associated with a second population of clusters with distinguishable structures, to be called form 2. The rest of the spectrum from this form is overlapped by that from form 1 so that only one g value, g3(2) = 2.003, is well-defined; the line width at this g value is -80 MHz. Before accepting the conclusion that there are two distinct cluster subpopulations, we examined the possibility that the total spectrum of the hydrogenasecluster can be simulated by applying a simple model developed by Guigliarelli et aL3 to describe the EPR spectra of the [3Fe4S]+ clusters. The model considers a trio of S = s / ferric ~ ions, each characterized by a g tensor and
TABLE II: Parameters for EPR !%ulationa According to Eqs3md4' [3F&]+ clusters uo AU e go1 go2 go3 A. uinclundii Fd I 0.095 0.018 0.2 2.032 2.032 2.032 D. gigas Fd I1 0.105 0.020 0.15 2.036 2.033 2.038 T.commune Fd (an) 0.106 0.020 0.16 2.042 2.028 2.038 D. uf~ccmurFd I11 0.130 0.025 0.30 2.038 2.041 2.011 D. gigus hydrogenad form 1 (70%) O.Oao(5) -ob 0.2 2.035 2.033 2.037
form 2 (30%) 0.077(1) 0.003 0.2 a D. gigushydrogenase, this work all others fromref 3. All calculations usc c = 0.1 (cq 4) (see text); UO. the most probable value of u D/Jand Au,the width of the distribution in u, are defined in text, as are the other parameters. Note that thegtensor axes (1,2,3) correspond to the ( x j y ) spin Hamiltonian axes, respectively. * More precisely. Au < 3 X l e .
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zero-field splitting (zfs) tensor. These ions are coupled by exchange interactions as first described by Kent et a1.12 where the exchange parameters are written, 312 = J, J23 = J( 1 c) and J23 = J( 1 d), with 0 < z < 3 / ~and d
