Atomic Resolution Structures of Rieske Iron-Sulfur
Protein: Role of Hydrogen Bonds in Tuning
the Redox Potential of Iron-Sulfur Clusters
Derrick J. Kolling,1Joseph S. Brunzelle,3SangMoon Lhee,1Antony R. Crofts,1,2and Satish K. Nair1,2,* 1
Center for Biophysics and Computational Biology 2
Department of Biochemistry, University of Illinois at Urbana-Champaign, 600 S. Mathews Avenue, Urbana, IL 61801, USA 3
Northwestern University Center for Synchrotron Research, Life Sciences Collaborative Access Team, Argonne, IL 60439, USA *Correspondence:[email protected]
DOI 10.1016/j.str.2006.11.012
SUMMARY
The Rieske [2Fe-2S] iron-sulfur protein of
cyto-chrome bc
1functions as the initial electron
ac-ceptor in the rate-limiting step of the catalytic
reaction. Prior studies have established roles
for a number of conserved residues that
hydro-gen bond to ligands of the [2Fe-2S] cluster. We
have constructed site-specific variants at two
of these residues, measured their
thermody-namic and functional properties, and
deter-mined atomic resolution X-ray crystal
struc-tures for the native protein at 1.2 A˚ resolution
and for five variants (Ser-154/Ala, Ser-154/
Thr, Ser-154/Cys, 156/Phe, and
Tyr-156/Trp) to resolutions between 1.5 A˚ and
1.1 A˚. These structures and complementary
biophysical data provide a molecular
frame-work for understanding the role hydrogen
bonds to the cluster play in tuning
thermody-namic properties, and hence the rate of this
bioenergetic reaction. These studies provide a
detailed structure-function dissection of the
role of hydrogen bonds in tuning the redox
potentials of [2Fe-2S] clusters.
INTRODUCTION
The ubihydroquinone:cytochrome c oxidoreductase (E.C. 1.10.2.2) (bc1 complex) plays a fundamental role in the
major biological energy-conservation pathways, including those of the mitochondrial and bacterial respiratory and photosynthetic chains (Crofts, 2004; Crofts and Berry, 1998). The enzyme catalyzes the transfer of electrons from quinol to a small soluble protein, such as cytochrome c, and couples the redox work to the generation of a proton gradient across the membrane (Crofts and Berry, 1998). The core components of this multisubunit, integral mem-brane enzyme consist of three catalytic subunits (cyto-chrome b; cyto(cyto-chrome c1; and the Rieske iron-sulfur
pro-tein, ISP) bearing four redox centers (two cytochrome
b hemes, one cytochrome c1heme, and a high-potential
Rieske-type [2Fe-2S] cluster) (Berry et al., 2000). The pro-ton-motive Q-cycle model (Crofts et al., 1983; Mitchell, 1976) accounts for the overall function, and more recent work has shown that the rate-limiting step in the reaction is the oxidation of quinol at the Qo site (Crofts and Wang, 1989). The rate limitation occurs in the transfer of the first electron to the high-potential ISP (Hong et al., 1999), from where it is transferred through to cytochrome
c1. The semiquinone species remaining at the Qosite is
oxidized upon transfer of the second electron to the lower-potential b-type heme (Brandt and Trumpower, 1994; Crofts and Meinhardt, 1982).
Although initial crystallographic studies of mitochon-drial cytochrome bc1 complex did not resolve the ISP,
they showed that the Rieske cluster could occur in differ-ent configurations (Xia et al., 1997). The first structures in which the ISP was resolved showed that the Rieske ISP extrinsic domain was in several different positions, leading to the suggestion that this domain undergoes movement during catalysis (Zhang et al., 1998). This movement is re-flected in at least eight different positions now observed in native structures in different crystal forms of the bc1
com-plex (Iwata et al., 1998; Zhang et al., 1998). Consistent with this hypothesis, mutants of the bc1 complex in which
movement of the Rieske ISP domain is constrained by in-tersubunit disulfide bridges showed a decrease in the rate of intramolecular electron transfer (Xiao et al., 2000). Although the movement of the Rieske ISP has been pro-posed to be a rate-limiting step in electron transfer, kinetic measurements after flash activation of the
Rhodo-bacter sphaeroides bc1complex in situ or in the isolated
complex demonstrate that rates associated with the movement of the Rieske ISP are much more rapid than the rate of ubiquinol oxidation (Crofts et al., 2003; Eng-strom et al., 2002).
Despite the low overall sequence homology between the mitochondrial and chloroplast bc1 complexes, the
crystal structures of their respective isolated Rieske ISP domains are quite similar, and residues that surround the [2Fe-2S] cluster are highly conserved (Carrell et al., 1997; Iwata et al., 1996). Most importantly, the protein ligands that engage the cluster are surrounded by a net-work of hydrogen bonds mediated by backbone amides
and residue side chains that are identical between the mi-tochondrial, bacterial, and chloroplast enzymes of the bc1
complex family, but vary in lower-potential Rieske-like proteins (Bonisch et al., 2002; Carrell et al., 1997; Hun-sicker-Wang et al., 2003; Iwata et al., 1996). In particular, the phenolic side chain of Tyr-156 (Rhodobacter number-ing) is located within hydrogen bond distance to the sulf-hydryl of cluster ligand residue Cys-129, and the hydroxyl of Ser-154 is proximal to one of the sulfur atoms in the cluster. Mutations of the residues equivalent to Tyr-156 in Rhodobacter sphaeroides (Guergova-Kuras et al., 2000), Saccharomyces cerevisiae (Denke et al., 1998),
Bos taurus (Leggate and Hirst, 2005), and Paracoccus
de-nitrificans (Schroter et al., 1998) are shown to affect the re-dox potential of the Rieske ISP. This hydrogen bond net-work is thought to increase the potential of the [2Fe-2S] cluster by charge delocalization to preferential stabiliza-tion of the reduced state.
Recently, we constructed mutant strains of R.
sphaer-oides expressing variant bc1 complexes in which these
residues have been replaced by site-directed mutagene-sis (Guergova-Kuras et al., 2000). Each of these mutant strains was able to assemble the bc1complex and support
photosynthetic growth. Redox titration of purified mutant
bc1 complexes demonstrates that the Tyr-156/Leu,
Tyr-156/Phe, and Tyr-156/Trp substitutions result in an alteration of the midpoint potential of the Rieske ISP and a decrease in the rate of quinol oxidation. Flash-induced kinetic measurements of bc1 complexes
in situ demonstrate that changes in the pK value of the oxidized Rieske ISP results in a change in the pH depen-dence of the rate of quinol oxidation (Guergova-Kuras et al., 2000).
In order to provide a context for further exploring the contribution of hydrogen bonds in tuning the redox po-tential of iron-sulfur clusters, we have characterized the kinetic properties and undertaken structural studies of the wild-type and site-specific variants of the isolated, water-soluble Rieske ISP domain from R. sphaeroides. The redox potential and pK values of these site-specific variants were measured by using protein film voltamme-try (Zu et al., 2003). In order to provide a molecular ba-sis for understanding these biophysical measurements, we have determined high-resolution X-ray crystal struc-tures of wild-type Rieske ISP and five mutants at clus-ter-proximal residues Ser-154/Ala, Ser-154/Thr, Ser-154/Cys, Tyr-156/Phe, and Tyr-156/Trp to res-olutions of better than 1.5 A˚. The high resolution of the structural data allows for unambiguous assignments of all atoms and solvent molecules as well as alternate side chain conformers. These structures allow for atomic-level dissection of biophysical results to demon-strate the specific role of protein ligands in tuning the electrochemical properties of the [2Fe-2S] clusters. Our studies demonstrate that observed decreases in midpoint potential that result from the mutation of resi-dues involved in hydrogen bonding with the cluster are a direct consequence of the removal of individual hydro-gen bonds.
RESULTS AND DISCUSSION Structure Determination
A highly redundant data set, collected at the Cu Ka wave-length, was used to determine the crystal structure of the water-soluble Tyr156/Phe Rieske ISP by single-wave-length anomalous diffraction methods. Despite the mod-est anomalous signal from Fe at this wavelength (f000 = 3.02 at 1.54 A˚), the quality of the resultant electron density map was excellent and allowed for the entire trace of the protein main chain with automated methods. Rapid man-ual building was facilitated by the excellent starting phase set, allowing for a straightforward trace of the entire poly-peptide and associated solvent molecules. The structures of wild-type, Ser156/Ala, Ser156/Thr, Ser154/Cys, and Tyr156/Trp Rieske ISPs were solved by molecular replacement by using the final refined coordinates of Tyr156/Phe Rieske and were refined to resolutions be-tween 1.1 A˚ and 1.5 A˚ resolution. The crystal structures of fully oxidized and fully reduced wild-type Rieske ISP were each determined to a resolution of 1.05 A˚; crystals soaked in a 5-fold molar excess of either sodium ascor-bate (reduced state) or potassium hexacyanoferrate (oxi-dized state) were used. All data collection and refinement statistics may be found inTable 1.
Overall Structure
The overall polypeptide fold is similar to that of isolated Rieske ISP from mitochondria and chloroplast, as well as to those of Rieske-type proteins of bacterial and ar-chaeal origins (Bonisch et al., 2002; Carrell et al., 1997; Hunsicker-Wang et al., 2003; Iwata et al., 1996). As de-tailed comparison of the high-resolution structures of ox-idized and reduced wild-type Rieske ISP did not show any significant differences between the structures (S.K.N., D.J.K., J.S.B., and A.R.C., unpublished data), the struc-tural details discussed below are applicable to both redox states. It should be noted that although crystals of the ox-idized form of the Rieske ISP were maintained in the pres-ence of excess oxidant, exposure to high flux-X radiation may have resulted in conversion into the reduced form.
The polypeptide consists of ten antiparallel b sheets lay-ered into three stacks (Figure 1). These secondary struc-ture elements are arranged to form two distinct entities, the cluster-binding domain and the basal domain. The first layer of b sheet is formed by strands b1, b11, and b12; the second layer is formed by strands b3–b5 and b10; and the third layer is formed by strands b6–b9. A single a helix adjoins b3 and b4 and connects the basal and cluster-binding domains. A disulfide bridge between two cysteine residues, located in the loop regions between b5-b6 (Cys-134) and b7-b8 (Cys-151), tethers the b5-b8 strand to-gether and provides further stability for loops that contain the cluster-binding residues. The basal domain of the
R. sphaeroides Rieske ISP is structurally similar to
equiv-alent domains of Rieske proteins from bovine, plant, and archaeal sources (Bonisch et al., 2002; Carrell et al., 1997; Colbert et al., 2000; Hunsicker-Wang et al., 2003; Iwata et al., 1996). The cluster-binding domain spans
50 residues within the center of the polypeptide se-quence and bears the cluster ligands and cluster-inter-acting residues.
Pairwise comparison of the structure of R. sphaeroides ISP with the isolated Rieske domains from other organ-isms reveals closest structural homology to the mitochon-drial Rieske ISP (root mean square deviation [rmsd] of 1.1 A˚ over 126 aligned residues; Z-score = 22.7) (Iwata et al., 1996). As studies of other Rieske ISPs have demon-strated relative reorientations of the basal and cluster-bind-ing domains (Hunsicker-Wang et al., 2003), a comparison of these individual domains is warranted. The cluster-binding domains of the R. sphaeroides and mitochondrial
ISPs show an rmsd of 0.4 A˚ over 48 aligned residues (75% identity), while the basal domains show an rmsd of 1.2 A˚ over 78 aligned residues (29% identity). The greatest deviation between the two basal domains occurs as a re-sult of a 13 amino acid loop insertion spanning Thr-96 through Ala-109 in the structure of the R. sphaeroides ISP. Structure-based comparisons with the isolated Rieske ISP from chloroplast demonstrate weaker homol-ogy (rmsd of 2.7 A˚ over 94 aligned residues; Z-score = 8.7; the cluster-binding domains show an rmsd of 1.0 A˚ over 45 aligned residues with 49% sequence identity; the basal domains show an rmsd of 2.6 A˚ over 47 aligned res-idues with 13% sequence identity) (Carrell et al., 1997). Table 1. Data Collection and Refinement Statistics
Wild-Type Tyr-156/Phe Tyr-156/Trp Ser-154/Ala Ser-154/Thr Ser-154/Cys Data Collection Statistics
Space group I4 I4 I4 I4 I4 I4
Beamline 32-ID Home 32-ID 22-ID 17-ID 22-ID
Cell lengths (A˚) a = 70.57, c = 54.77 a = 70.63, c = 54.78 a = 70.64, c = 54.60 a = 69.94, c = 54.58 a = 70.65, c = 54.72 a = 70.88, c = 54.93 Resolution (A˚) 1.2 1.5 1.1 1.35 1.5 1.5 Total observations 171,725 304,203 289,260 170,255 78,492 84,468 Unique reflections 41,254 21,562 54,430 29,270 20,919 21,968 Completeness (%) 98.0 (99.7) 99.7 (97.0) 99.8 (99.7) 100 (100) 96.7 (87.7) 99.9 (100) Redundancy 4.2 14.1 5.3 5.8 3.8 3.9 Rmerge(%) 3.0 (6.5) a 5.1 (18.3)a 5.0 (13.0)a 5.8 (47.8)a 5.6 (19.9)a 8.7 (45.3)a I/s(I) 24.7 (19.6)a 67 (15)a 16.7 (12.0)a 26 (3.8)a 21 (8)a 14.9 (3.5)
SAD analysis Tyr-156/Phe
Wavelength (A˚) 1.54
Mean FOM 0.58
Density-modified FOM 0.75 Refinement Statistics
Resolution bins (A˚) 12–1.2 18–1.5 13–1.2 18–1.35 18–1.5 18–1.5
Reflections used 38,022 19,979 51,606 27,466 19,380 20,692 Rcrystal 11.66 15.20 12.56 14.20 13.35 13.16 Rfree 13.37 16.72 13.74 15.92 17.37 17.85 Number of atoms Protein 1,088 1,065 1,123 1,065 1,079 1,062 Solvent 198 113 232 149 181 179 Glycerol 0 0 12 12 12 12 Average B (A˚2) 8.66 19.33 9.25 21 13.14 16.45
Rmsd bond length (A˚) 0.007 0.006 0.009 0.007 0.008 0.009
Rmsd bond angle () 1.508 1.334 1.476 1.388 1.406 1.462
PDB accession code 2NUK 2NUM 2NWF 2NVG 2NVE 2NVF
Rmerge= SjI hIij/SI, where I is the integrated intensity of a given reflection.
Rcrystal= SjjFoj jFcjj/SjFoj. Rfreewas calculated with 5%–7% of the data excluded from refinement. aNumbers in parentheses correspond to values for the outermost shell.
Hence, despite the overall weak sequence similarity, structural determinants between the R. sphaeroides and chloroplast ISPs are quite similar.
The [2Fe-2S] cluster is engaged by protein ligands Cys-129 (Sg-Fe distance of 2.33 A˚), His-131 (Nd1-Fe distance of 2.11 A˚), Cys-149 (Sg-Fe distance of 2.29 A˚), and His-152 (Nd1-Fe distance of 2.10 A˚). The exceptionally high resolution of 1.2 A˚ for the wild-type ISP structure provides an accurate limit to the metal-ligand distances. Full-matrix, least-squares refinement yields estimated standard devia-tions in the range of 0.01 A˚ for the Fe-S bond lengths and 0.02 A˚ for the Fe-N bond lengths. A detailed list of the clus-ter-ligand distances for wild-type and site-specific mutant
R. sphaeroides Rieske ISPs can be found inTable 2.
Role of Cluster-Proximal Residue Tyr-156
In addition to the [2Fe-2S] cluster and its associated li-gands, the local environment around the clusters of bc1
Rieske domains are characterized by a disulfide bridge formed between Cys-134 and Cys-151, and by two polar residues, Ser-154 and Tyr-156. Tyr-156 makes a hydrogen bond with the cluster ligand Cys-129 (Oh-Sg distance of 3.1 A˚) (Figure 2A), and Ser-154 is within hydrogen bond distance to atom S1 of the cluster (S1-Og distance of
3.2 A˚) (Figure 3A). Biochemical and biophysical studies establish that these hydrogen bonds can affect the redox potential and pKox1of the Rieske ISP (seeTable 3). For
ex-ample, the Tyr-156/Phe mutation in the R. sphaeroides Rieske ISP results in a 45 mV decrease in the midpoint po-tential relative to the wild-type (Table 3). Similar results
were obtained in S. cerevisiae and P. denitrificans, in which the equivalent Tyr/Phe substitution results in a decrease in the midpoint potential of the ISP by 65 and 44 mV, respectively. In the absence of structural data, it was unclear if these changes in the midpoint potential were solely the consequence of a loss of a hydrogen bond between Tyr-156 and Cys-129.
Our 1.5 A˚ resolution crystal structure of the Tyr-156/ Phe R. sphaeroides Rieske ISP reveals that the structure of the mutant is nearly identical to that of the wild-type ISP. Overall, the crystal structures of the two ISPs can be superimposed with a rmsd of 0.105 A˚, and there are no local alterations in the polypeptide scaffold that result from the Tyr-156/Phe mutation. Analysis of electron density maps calculated with either experimental phases or phases from the coordinates of the final refined struc-ture do not show the presence of any additional feastruc-tures in the vicinity of the Phe-156 side chain (Figure 2B). Most importantly, there is no evidence of any ordered sol-vent molecules that may compensate for the loss of the hydroxyl group in the Phe-156 mutant. Hence, the mea-sured 45 mV decrease in the midpoint potential in the Tyr-156/Phe R. sphaeroides ISP, relative to the wild-type, is solely the consequence of the loss of the hydrogen bond between Phe-156 and the cluster ligand Cys-129.
In contrast to the modest changes in both structure and biophysical characteristics that accompany the Tyr-156/ Phe mutation, significant alterations are observed in our 1.1 A˚ structure of the Tyr-156/Trp mutant R. sphaeroides ISP (Figure 2C). Although the overall structure of this Figure 1. Orthogonal Views of the Over-all Structure of the Rieske Iron-Sulfur Protein from R. sphaeroides
(A) A ribbon representation of the polypeptide is shown and is labeled with the corresponding secondary structural elements as defined in the text. Atoms within the [2Fe-2S] cluster are shown as white and yellow spheres. (B) The structure shown is rotated 90about
the vertical axis and is viewed at the location of the [2Fe-2S] cluster.
Table 2. Atomic Distances, in A˚ , between [2Fe-2S] Cluster Residues and Protein Ligands of Interest
Variant C129-Fe1 H131-Fe2 C149-Fe1 H152-Fe2 Y156-C129 S154-S1
Wild-type 2.33 2.11 2.29 2.10 3.14 3.17 S154A 2.33 2.06 2.31 2.12 3.11 N/A S154C 2.35 2.18 2.31 2.16 3.13 3.07 S154T 2.33 2.12 2.30 2.12 3.12 3.16 Y156F 2.31 2.14 2.32 2.14 N/A 3.18 Y156W 2.32 2.12 2.29 2.10 N/A 3.18
variant is similar to that of the wild-type ISP (rmsd of main chain atoms of 0.124 A˚), accommodation of the indole ring of Trp-156 requires movement of the polypeptide back-bone at regions adjacent to this residue. Specifically, the main chain along two loop regions that flank Trp156, span-ning residues Gly-127 through Ile-137 and Gly-146 through Gly-153, are pushed apart0.3–0.4 A˚ (Figure 2D). As all of the cluster ligands are harbored within this region, this movement results in a slight shift of the entire cluster by 0.4 A˚ relative to its position in the wild-type ISP. However, despite the structural perturbation, the integrity of the hy-drogen bond between Ser-154 and atom S1of the cluster
is not compromised (S1-Og distance of 3.2 A˚).
Prior studies from our laboratory have shown that the Tyr-156/Trp mutation in the R. sphaeroides ISP results in a decrease in the midpoint potential by 100 mV, a change in pKox1by0.9 units, and substantial changes
in the EPR spectra relative to the wild-type ISP ( Guergova-Kuras et al., 2000). However, despite the modification in the EPR spectra, this mutant retained the characteristic Rieske-type signal and displayed sensitivity to the inhibi-tor stigmatellin, similar to that of wild-type ISP. Our 1.1 A˚ resolution crystal structure provides a structural rationale for understanding these biophysical characteristics of the Tyr-156/Trp mutant R. sphaeroides ISP. The structural rearrangements noted above, which are required to ac-commodate the Tyr-156/Trp mutation, result in pertur-bations of the EPR spectra of this variant relative to that of the wild-type ISP as well as a greater perturbation of Figure 2. Close-Up View of Mutations at Residue 156
(A–C) Difference Fourier maps shown around residue 156 of wild-type and variant Rieske ISPs, calculated with coefficients Fo Fcby using
experimental amplitudes and phases calculated from the final refined structures, minus the coordinates of residue 156. The contour levels of the maps are 2.5s (blue). Coordinates from the final refined structures are superimposed on the respective maps, and atoms of the [2Fe-2S] cluster are shown as stick figures in magenta and orange. The struc-tures shown are of (A) wild-type, (B) 156/Phe, and (C) Tyr-156/Trp Rieske ISPs.
(D) Superposition of the structures of wild-type (cyan) and Tyr-156/ Trp (yellow) ISPs showing the gross structural movements that accom-pany the introduction of the Trp-156 side chain.
Figure 3. Close-Up View of Mutations at Residue 154
(A–D) Difference Fourier maps shown around residue 154 of wild-type and variant Rieske ISPs, calculated with coefficients Fo Fcby using
experimental amplitudes and phases calculated from the final refined structures, minus the coordinates of residue 154. The contour levels of the maps are 2.5s (blue) and 8s (red). Coordinates from the final re-fined structures are superimposed on the respective maps, and atoms of the [2Fe-2S] cluster are shown as stick figures in magenta and or-ange. The structures shown are of (A) wild-type, (B) Ser-154/Ala, (C) Ser-154/Cys, and (D) Ser-154/Thr Rieske ISPs.
the thermodynamic properties than in the Tyr-156/Phe mutation.
Role of Cluster-Proximal Residue Ser-154
In the wild-type structure of R. sphaeroides ISP, Ser-154 is within hydrogen bond distance of the bridging sulfur atom, S1, of the cluster (S1-Og distance of 3.2 A˚) (Figure 3A). In
order to determine the contribution of this residue in mod-ulating redox potential, we have characterized kinetic pa-rameters of site-specific variants at this residue and deter-mined high-resolution crystal structures of the variant ISPs. Removal of the hydroxyl group in the Ser-154/ Ala mutation results in a decrease in the midpoint potential by 97 mV, an increase in pKox1to 8.02, a 10-fold decrease
in the association constant for formation of the ES com-plex with quinol, and a 5-fold decrease in sensitivity to inhibition by stigmatellin (Table 4). The structure of Ser-154/Ala R. sphaeroides ISP has been determined to
a resolution of 1.35 A˚. A comparison of a least-squares su-perposition of the wild-type and Ser-154/Ala ISP struc-tures (rmsd of main chain atoms of 0.134) shows that min-imal structural perturbations result as a consequence of the mutation. No additional features, corresponding to ordered solvents, can be observed in the vicinity of the cavity created by the Ser-154/Ala mutation (Figure 3B). Consequently, the measured changes in kinetic parame-ters in the Ser-154/Ala mutant ISP can be attributed to be a direct consequence of the loss of the hydrogen bond between Ser-154 and a bridging sulfur atom in the [2Fe-2S] cluster.
The 1.5 A˚ crystal structure of the Ser-154/Thr
R. sphaeroides ISP is nearly identical to that of the
wild-type ISP (rmsd of main chain atoms of 0.141 A˚) and reveals no local or global changes in the polypeptide that result from the mutation. The hydroxyl of Thr-154 occupies the same location as that of Ser-154 in the wild-type ISP Table 3. Biochemical and Biophysical Parameters for ISP Variants
Variant DpH Optimuma Method E m, 7.4Noteb Eacid (mV) Ealk
(mV) pKox1 pKox2 pKred1 pKred2 Reference
R. sphaeroides
Wild-typec — PFV 297 308
134 7.60 9.60 12.4 12.4 (Zu et al., 2003)
Wild-typed — CD 283 (Ugulava and
Crofts, 1998) S154Tc 0.3 PFV 274 283 168 7.70 9.28 12.37 12.37 f S154Td 0.3 CD 255f S154Cc 0.5 PFV 295 319 107 7.14 9.73 12.10 12.10 f S154Cd 0.5 CD 300f S154Ac 0.3 PFV 170 173 307 8.02 9.92 13.10 13.10 f S154Ad 0.3 CD 186f Y156F 0.2 CD 238 256 <56 7.5 9.2 >10 >10 (Guergova-Kuras et al., 2000) Y156W 1.2 CD 198 198 <114 8.5 >10 >10 >10 (Guergova-Kuras et al., 2000) Bovine Wild-typee PFV 311 152 7.55 9.10 11.80 12.81 (Leggate and Hirst, 2005) Wild-typec CD 315 7.70 9.10 (Link, 1999)
S163Ae PFV 164 297 8.15 9.31 11.88 13.54 (Leggate and
Hirst, 2005) Y165Fe PFV 252 225 7.74 9.43 12.08 13.31 (Leggate and Hirst, 2005) Y165We PFV 214 <216 8.00 9.40 >12.40 >12.40 (Leggate and Hirst, 2005) apH optimum for turnover of the Q
osite was at pH 7.6 and was measured at Eh100 mV inUgulava and Crofts (1998). It was at pH 7.2 and was measured at Eh70 mV in current work.
bIn the intact bc
1complex, formation of the gx= 1.800 complex with ubiquinone shifts the apparent Em. c
Rieske iron-sulfur protein (ISP). d
Measured in situ in the bc1complex. e
Recombinant ISP protein expressed in E. coli. f
structure and donates a hydrogen bond to atom S1of the
cluster (S1-Og distance of 3.2 A˚) (Figure 3C). The Cg of
Thr-154 protrudes into a hydrophobic cleft defined by Ile-162 and Tyr-156 and is accommodated in this cleft by movement of Cd of Ile-162, with minimal changes in the position of neighboring atoms. The Ser-154/Thr mu-tation results in only moderate alteration in kinetic param-eters, with a 28 mV decrease in midpoint potential, and a modest increase in the association constant for interac-tion with quinone. As Thr-154 donates a hydrogen bond to atom S1 of the cluster (S1-Og distance of 3.2 A˚), the
changes in the kinetic parameters observed in the Ser-154/Thr mutation, relative to wild-type ISP, are relatively modest.
The Ser-154/Cys mutation results in a 17 mV increase in the midpoint potential relative to the wild-type ISP. The 1.5 A˚ crystal structure of the Ser-154/Cys R.
sphaer-oides ISP shows no significant changes in the polypeptide
structure as a consequence of the mutation relative to the wild-type ISP (rmsd of main chain atoms of 0.128 A˚). Com-parison of the wild-type and Ser-154/Cys mutant ISP reveals that the side chain of Cys-154 is rotated about tor-sion angle c1. As a result, the Sg of Cys-154 occupies a
position closer to atom S1of the [2Fe-2S] cluster (S1-Og
distance of 2.9 A˚). The increase in midpoint potential in this mutant relative to the wild-type enzyme may be a result of the decrease in the interatomic distance to the [2Fe-2S] cluster, which would result in a stronger hydro-gen bond between Cys-154 and the bridging sulfur. Presumably, formation of a disulfide between Cys-154 and the bridging sulfur is precluded by the cluster micro-environment.
Conclusions
The importance of hydrogen bonds in determining the re-dox potential of [2Fe-2S] clusters has long been recog-nized (Adman et al., 1975; Carter, 1977). Earlier attempts to dissect the contributions of hydrogen bonding to redox potentials utilized spectroscopic and structural studies of [4Fe-4S] clusters from different proteins (Backes et al., 1991). However, electrostatic calculations demonstrate that the observed variations in redox potentials amongst these different proteins are not due solely to differences
in hydrogen-bonding patterns, as cluster microenviron-ment contributes significantly to the redox potential ( Lan-gen et al., 1992). More recent efforts to analyze the contri-bution of protein ligands to the tuning of iron-sulfur cluster redox potentials have focused on mutational analyses of Rieske [2Fe-2S] proteins (Denke et al., 1998; Leggate and Hirst, 2005; Schroter et al., 1998). Rieske ISPs are ideal candidates for such analysis due to (1) their higher re-dox potential compared to other [2Fe-2S] proteins, and (2) the complex hydrogen-bonding network surrounding the cluster, which facilitates mutational analyses without se-verely compromising structural integrity (Carrell et al., 1997; Iwata et al., 1996). However, mutational studies are incomplete in the absence of high-resolution structural information to provide a framework for understanding the structural consequences. Our spectroscopic, kinetic, and crystallographic analyses of site-specific mutants of the
R. sphaeroides Rieske ISP represent the first, to our
knowledge, detailed structure-function dissection of the role of hydrogen bonds in tuning the redox potentials of [2Fe-2S] clusters. These studies demonstrate that the ob-served decreases in midpoint potential that result from the mutation of residues involved in hydrogen bonding with the cluster are a direct consequence of the removal of in-dividual hydrogen bonds. The Rieske ISP is able to toler-ate modest changes in the nature of the hydrogen-bond-ing ligand with minimal effects on either the structure or the redox potential of the [2Fe-2S] cluster. Finally, our studies demonstrate that the ISP is sufficiently plastic to accommodate introduction of large changes into the prox-imity of the cluster with only slight alterations in the protein scaffold.
EXPERIMENTAL PROCEDURES Rieske Protein Production
Details on the construction of Rhodobacter sphaeroides strains ex-pressing mutant Rieske ISPs have already been described ( Guer-gova-Kuras et al., 2000). Briefly, mutations in the ISP were made by using the QuikChange kit (Stratagene) for site-directed mutagenesis, with minor modifications. In order to express the desired mutant pro-teins, the DNA fragment containing a mutation site was cloned in a broad host plasmid, pRK415, and transferred into host strain R.
sphaeroides BC17 from which the chromosomal fbc operon encoding
Table 4. Kinetic Parameters for FS154 Mutants
Strain Emof the ISP (mV)a cyt bHReduction Ratesb Percentage of Reduction Rates Apparent Emof the cyt bHReduction
Association Constant for QH2(KQH2)c Stigmatellin Inhibitiond Wild-typee 283 ± 5 1370 100 129.4 ± 2.4 23.5 0.585 FS154T 255 ± 5 669 48.7 134.5 ± 1.9 35.3 2.39 FS154C 300 ± 4 871 63.4 119.2 ± 2.9 10.4 2.30 FS154A 186 ± 5 46.7 3.40 101.5 ± 2.1 2.52 2.57 a
The Emvalues are measured at pH 7.4 by CD spectroscopy. b
When the quinone pool is30% reduced (mol. cyt bH reduced
3[mol. total bc1]13s1). c
The values are derived as suggested byZhang et al. (1998). d
The amount of inhibitor that blocked half of the functional bc1complex (mol. stigmatellin/mol. bc1complex). e
the bc1complex had been deleted (Yun et al., 1990). The integrity of
each mutation was confirmed at the DNA level by sequencing of the plasmid purified from the cells from which the protein was purified. Wild-type and mutant cytochrome bc1complexes were purified with
nickel-affinity resin by virtue of an engineered carboxy-terminal poly-histidine tag (Guergova-Kuras et al., 1999).
Preparation of a Soluble Rieske Fragment
After purification of the multisubunit complex, the soluble Rieske sub-unit extrinsic domain was liberated by using thermolysin digestion and was further purified by hydrophobic interaction chromatography. Mass spectrometric analysis documents that the soluble domain spans residues 47–196 of the full-length subunit. Purified samples were supplemented with glycerol and stored at 193K.
Preparation of the Intact Rieske Subunit
For protein film voltammetry, the intact subunit was isolated from the cytochrome bc1complex with retention of the hydrophobic N-terminal
domain. The cytochrome bc1complex was prepared as described
previously (Guergova-Kuras et al., 1999). For fractionation, it was exchanged into a solution containing 10 mM CAPS and 0.01%
n-decyl-B-D-maltoside (pH 12), incubated on ice for 30 min, and fil-tered with a Centriplus centrifugal concentrator with a cutoff of 50 kDa (Amicon, Beverly, MA). The Rieske protein was contained in the filtrate. It was exchanged into 20 mM histidine (pH 7.5), 0.5 mM EDTA, 1% glycerol (vol/vol), and 500 mM KCl and was concentrated with a 7 kDa cutoff concentrator.
Protein Film Voltammetry
Reduction potentials were measured by using protein film voltammetry essentially as described previously (Zu et al., 2002a, 2002b). It was found that the intact Rieske subunit provided better adhesion to the electrode surface, and this preparation was used for most experi-ments. Briefly, the protein was applied directly to a freshly polished py-rolytic graphite-edge (PGE) electrode surface and was then placed into solution in an all-glass cell. The cell was thermostated and encased in a Faraday cage. Measurements were performed aerobically, but this was not a problem as oxygen did not interfere with measurements in the range of 100–500 mV (versus SHE). Analog-scan cyclic voltamme-try was performed with a Bioanalytical Systems (West Lafayette, IN) CV-27 Voltammograph with an in-house amplifier, and results were re-corded via an in-house program. Data were analyzed by using Fourier transformation and cubic-spline subtraction in Origin 6.1 (OriginLab Corp.; Northampton, MA). Solution pH values were controlled by using mixtures of four of the following buffers depending on the pH (total concentration, 40 mM): 10 mM sodium acetate, HEPES, MES, TAPS, CAPS, and sodium phosphate. Volumetric solutions of NaOH were used above pH 13. All standard reagents were supplied by Fluka (Buchs, Switzerland) or Merck-BDH (Poole, UK). The pH of each solu-tion was checked immediately after measurement; 1 M NaOH was used as a pH 14 standard. The effects of high Na+
concentration were corrected by using standard formulae (Bard and Faulkner, 2001).
Crystallization
Samples of the soluble Rieske ISP extrinsic domain were thawed and exchanged into buffer consisting of 100 mM KCl, 20 mM HEPES (pH 7.5) through ultrafiltration. Initial crystallization conditions were estab-lished by the sparse-matrix sampling methods with commercial and homemade screens. Refinement of promising conditions yielded large, red-colored crystals suitable for diffraction analysis. Crystals of the Rieske ISP were grown by using the hanging-drop vapor-diffu-sion method. Briefly, 2 ml protein sample (8 mg/ml) was added to 2 ml precipitant (1.5 M [NH4]2SO4, 100 mM sodium acetate [pH 4.6], and
20% [v/v] anhydrous glycerol) and equilibrated over a well containing the precipitant solution at 20C. Crystals grew in 24 hr and reached
a maximum size of 0.5 3 0.5 3 0.8 mm in 3 days. Crystals of mutant Rieske ISP were grown under similar conditions and grew to the same size. Crystals of fully oxidized and fully reduced wild-type Rieske ISP were produced by soaking preformed crystals in a 5-fold molar ex-cess of either sodium ascorbate (reduced state) or potassium
hexa-cyanoferrate (oxidized state). Despite the presence of suitable concen-trations of a cryopreservant in the crystallization buffer, diffraction data from crystals harvested directly from the crystallization drop were hampered by the presence of ice rings. To circumvent this problem, crystals of Rieske ISP were transiently streaked through a solution of Paratone-N immediately prior to flash cooling by direct submersion in liquid nitrogen.
Data Collection and Structure Determination
Crystals of Rieske ISP diffracted to better than 1.5 A˚ with a Raxis IV++ image plate detector mounted on a Cu Ka Rigaku R3H home source rotating anode generator. These crystals occupied space group I4 with unit cell parameters a = b = 70.63 A˚, c = 54.78 A˚. A data set en-compassing a complete 360sweep (1oscillations) was collected
from a single crystal of the Tyr156/Phe mutant Rieske ISP. This data set had an overall redundancy of 15-fold including all data and 7-fold with anomalous pairs kept separate. Data were integrated and scaled with the HKL2000 package (Otwinowski and Minor, 1997).
The high-symmetry space group occupied by these crystals allowed for the simultaneous measurement of a significant number of Bijvoet pairs and, coupled with the high quality of the data (seeTable 2), pro-duced a strong anomalous signal from the [2Fe-2S] cluster at the wavelength of Cu Ka. The position of the two Fe peaks were readily identified by using SHELXC, and SHELXD/SHELXE were used to ex-tract phases from these heavy atom sites (Schneider and Sheldrick, 2002). The resultant phases were density modified with RESOLVE (Terwilliger, 2003), and the modified phases were imported into ARP/ wARP version 5.1 (Morris et al., 2003) for automated chain tracing. Au-totracing resulted in a complete trace of the protein main chain and as-signment of more than half of the side chains of the protein sequence. The remainder of the model was built with XtalView (McRee, 1999) and further improved through rounds of refinement with REFMAC5 ( Mur-shudov et al., 1997; Vagin et al., 2004) and manual rebuilding. Cross-validation, with 7% of the data for the calculation of Rfree, was utilized
to monitor building bias (Brunger, 1993).
Data from crystals of wild-type, Ser156/Ala, Ser156/Thr, Ser156/Cys, and Tyr156/Trp Rieske ISPs were collected to limiting resolutions between 1.5 and 1.1 A˚ at an insertion device synchrotron source (Sector 32 ID, Sector 17 ID, and Sector 22 ID, Advanced Photon Source, Argonne, IL) by using charge-coupled device detectors. Phases for each structure were determined by molecular replacement with the program MOLREP (Vagin and Teplyakov, 2000) by using the Tyr156/Phe structure as a probe. Multiple rounds of manual model building were interspersed with refinement by using REFMAC5 (Murshudov et al., 1997; Vagin et al., 2004) to complete structure re-finement. Alternate conformers were modeled where appropriate. Individual hydrogen atoms were included as riding atoms in the high-est-resolution structures (1.2 A˚ or better) based on features in differ-ence Fourier maps. Cross validation used 5%–7% of the data in the calculation of Rfree. Graphics were created by using Ribbons (Carson, 1996) and Pymol (pymol.sourceforge.net).
ACKNOWLEDGMENTS
We thank Drs. Lisa Keefe and Irina Koshelev for support at Beamline 17-ID (IMCA-CAT), Drs. Marie Graham and Zhongmin Jin for support at Beamline 22-ID (SER-CAT), and Dr. Zdzislaw Wawrzak for support at Beamline 5-ID (DND-CAT); all researchers are at Argonne National Labs. We thank Denise Lorenz, Conor Danstrom, and Nathan Wetter for initial crystallization efforts. A.R.C. thanks Judy Hirst for advice in setting up the protein film voltammetry apparatus. S.K.N. thanks Dr. Colin Wraight for his continued support of structural biology at the University of Illinois at Urbana-Champaign (UIUC). This work was supported by grants from the American Cancer Society and UIUC (S.K.N.) and the National Institutes of Health GM35438 (A.R.C.). S.K.N. and A.R.C. designed research. D.J.K., SM.L., J.S.B., and S.K.N. performed research. D.J.K., SM.L., A.R.C., and S.K.N. analyzed data.
S.K.N. wrote the paper with input from D.J.K. and A.R.C. The authors declare no competing interests.
Received: August 8, 2006 Revised: November 16, 2006 Accepted: November 16, 2006 Published: January 16, 2007
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Accession Numbers
Coordinates have been deposited in the Protein Data Bank (http:// www.rcsb.org) with accession codes 2NUK (wild-type), 2NUM
(Y156F),2NWF(Y156W),2NVE(S154T),2NVF(S154C), and2NVG
