1 TITLE: Structural basis of interprotein electron transfer in bacterial sulfite
2 oxidation.
3 AUTHORS: Aaron P. McGratha,1, Elise M. Lamingb,2, G. Patricia Casas Garciac,
4 Marc Kvansakulc, J. Mitchell Gussb, Jill Trewhellab, Benoit Calmesd,e, Paul V.
5 Bernhardtd,e, Graeme R. Hansond,f,3, Ulrike Kapplerd,e,4, Megan J. Maherb,4
6
7 AFFILIATIONS:
8 aStructural Biology Program, Centenary Institute, Locked Bag 6, NSW 2042,
9 Australia
10 bSchool of Molecular Bioscience, University of Sydney, NSW, 2006, Australia.
11 cLa Trobe Institute for Molecular Science, La Trobe University, Melbourne, 3086,
12 Australia.
13 dCentre for Metals in Biology, eSchool of Chemistry and Molecular Biosciences and
14 fCentre for Advanced Imaging, The University of Queensland, Brisbane, QLD 4072,
15 Australia.
16 1Present address: Skaggs School of Pharmacy and Pharmaceutical Sciences,
17 University of California at San Diego, La Jolla, CA, 92037, USA.
18 2Present address: The Victor Chang Cardiac Research Institute, Sydney, NSW, 2010,
19 Australia.
20 3Deceased
21 4Correspondence: [email protected] (MJM)
22 [email protected] (UK)
23 COMPETING INTERESTS: The authors declare no financial or non-financial 24 competing interests.
1 25 ABSTRACT
26 Interprotein electron transfer underpins the essential processes of life and relies on the
27 formation of specific, yet transient protein-protein interactions. In biological systems,
28 the detoxification of sulfite is catalyzed by the sulfite-oxidizing enzymes (SOEs),
29 which interact with an electron acceptor for catalytic turnover. Here, we report the
30 structural and functional analyses of the SOE SorT from Sinorhizobium meliloti and
31 its cognate electron acceptor SorU. Kinetic and thermodynamic analyses of the
32 SorT/SorU interaction showed the complex is dynamic in solution, and that the
33 proteins interact with Kd = 13.5 ± 0.8 μM. The crystal structures of the oxidized SorT
34 and SorU both in isolation and in complex, reveal the interface to be remarkably
35 electrostatic, with an unusually large number of direct hydrogen bonding interactions.
36 The assembly of the complex is accompanied by an adjustment in the structure of
37 SorU and conformational sampling provides a mechanism for dissociation of the
38 SorT/SorU assembly.
39
2 40 INTRODUCTION
41 Although electron transfer reactions are key biochemical events, which underpin
42 fundamental processes, such as respiration and photosynthesis, the study of the
43 molecular details of the interprotein interactions at their core can be largely
44 intractable. In particular, atomic resolution crystal structures of electron transfer
45 complexes rare, due to their fundamentally transient nature (1). Electron transfer
46 pathways are made up of chains of redox proteins, which provide a path for the
47 controlled flow of electrons and rely on efficient docking of protein redox partners
48 through noncovalent, dynamic protein-protein interfaces (2). Complementary
49 electrostatic surfaces, hydrophobic interactions and dynamics at the protein-protein
50 interface have all been proposed to contribute to efficient interprotein electron transfer
51 (19), with a strong correlation between the driving force for the reaction, the distance
52 between redox centers and the rate of electron transfer (2, 20).
53
54 Interprotein electron transfer processes are central to the redox conversions of cellular
55 sulfur compounds, which are an evolutionarily ancient type of metabolism that has
56 existed as long as cellular life (3, 4). Sulfur-containing compounds mediate many
57 crucial reactions in the cell (for example, in coenzyme A, sulfur containing amino
58 acids or glutathione), but their reactivity also makes them potentially toxic (5). Sulfite
59 in particular, is a highly reactive sulfur compound that can cause damage to proteins,
60 DNA and lipids, resulting in oxidative stress and irreversible cellular damage (6). In
61 most cells, the detoxification of sulfite by oxidation to sulfate (Eq.(1)) is catalyzed by
62 sulfite oxidizing enzymes (SOEs) (7).
2- 2- + - 63 SO3 + H2O SO4 + 2H + 2e (1)
3 64 SOEs from plants, higher animals and bacteria have been characterized and they all
65 catalyze the same fundamental reaction. However, their cellular functions, catalytic
66 properties (5, 6, 8-10) and the identities of their natural electron acceptors vary
67 significantly. Some SOEs transfer electrons to oxygen (11), while others interact with
68 redox proteins such as cytochrome c (7, 8, 12-16) or as yet unknown cellular
69 components (5, 12, 13, 17). To date, three unique crystal structures of SOEs have
70 been reported: from chicken, plant and bacteria, which differ significantly in their
71 domain architectures and redox cofactor compositions. However, none of these
72 studies show details of a SOE in complex with its external electron acceptor (7, 11,
73 21). At present, no structural information on the molecular interactions of any of these
74 enzymes with their respective electron acceptors is available and the determinants that
75 dictate the type of electron acceptor individual SOEs employ, while maintaining the
76 efficiency of the basic enzyme reaction are open questions. (5, 18)
77
78 Here, we have investigated an electron transfer complex involving the periplasmic
79 SorT sulfite dehydrogenase from the α-Proteobacterium Sinorhizobium meliloti,
80 which represents a structurally uncharacterized type of SOE, and its electron acceptor,
81 the c-type cytochrome SorU (12, 17). In S. meliloti the SorT sulfite dehydrogenase is
82 part of a sulfite detoxification system that is induced in response to the degradation of
83 sulfur containing substrates such as organosulfonate taurine (17). Electrons derived
84 from sulfite oxidation are passed on to the SorU cytochrome, and likely then to
85 cytochrome oxidase, as S. meliloti is capable of sulfite respiration (12). Here we
86 report the crystal structures of both the isolated SorT and SorU proteins and the
87 biochemical and structural analyses of the SorT/SorU electron transfer complex. This
4 88 is the first time that a crystal structure of a molybdenum enzyme in complex with its
89 external electron acceptor has been solved.
90
91 RESULTS 92
93 The interactions between SorT and SorU are highly dynamic and efficient in
94 solution.
95 Sulfite-oxidizing enzymes, particularly those from bacteria, are known to be highly
96 efficient catalysts (5, 22). Previous work has established that SorT is able to transfer
97 electrons to the SorU cytochrome that is encoded on the same operon, however no
98 kinetic details of the interaction were reported (12). With the artificial electron
99 acceptor ferricyanide, SorT was shown to have turnover number of 338 ± 3 s-1 (12,
100 17). Employing SorU as the substrate, we analyzed the kinetics of the interaction
101 between SorT and SorU and found the interaction to be fast and highly specific, with
-1 102 a KM(SorU) of 32 ± 5 μM and a kcat of 140 ± 11 s , confirming that SorU is the natural
103 electron acceptor of SorT. Measurement of the thermodynamics of the SorT/SorU
104 interaction by isothermal titration calorimetry (ITC) revealed a dissociation constant
105 of Kd = 13.5 ± 0.8 μM with a determined stoichiometry of 0.8 ± 0.2. These values are
106 in the range observed for other electron transfer complexes (23, 24) and match a
107 model where SorT sequentially transfers two electrons, derived from sulfite oxidation,
108 to two SorU molecules. In other words, the SorT/SorU complex must form twice
109 (with two different ferric SorU protein molecules) to complete the oxidative half
110 reaction of SorT.
111
112 The KM of SorT for SorU is very close to its affinity for sulfite (KM=15.5 ± 1.9 μM
113 (17)), and the turnover number in the SorU-based assay is approx. 40% of that seen
5 114 with ferricyanide as the electron acceptor (17), where no significant reorientation and
115 docking of the electron acceptor is required. These data reveal interesting details
116 about the formation of the SorT/SorU complex, which appears to form with similar
117 affinities between the two proteins when both are oxidized as in the ITC experiments
118 and in a system where SorT constantly undergoes oxidation and reduction (SorU-
119 based enzyme assay). In addition, the similarity between the determined values of Kd
120 and KM indicates that the affinity of SorT for SorU is unaffected by the presence of
121 substrate or product. However, the kinetic parameters for this interaction are clearly
122 distinct from those for other sulfite-oxidizing enzymes, such as the bacterial SorAB
123 enzyme or chicken sulfite oxidase (CSO), both of which have much higher affinities
124 for their respective electron accepting cytochromes c (both with KM(Cyt c) ca. 2 μM)
-1 125 (25). The catalytic turnover of CSO is relatively slow (kcat = 47.5 ± 1.9 s ), due to a
126 mechanism requiring internal rearrangement, while turnover of SorAB with its natural
127 electron acceptor (334 ± 11 s-1) is significantly faster than that for SorT (22, 25).
128
129 The crystal structure of the SorT homodimer reveals a head to tail subunit
130 arrangement
131 In order to investigate whether there are any structural reasons for these differences,
132 we solved the crystal structure of SorT by molecular replacement and refined it to 2.4
133 Å resolution. The structure shows two homodimeric assemblies per asymmetric unit
134 (Table 1), which is in agreement with the quaternary structure as determined by
135 MALLS (12, 17). Unexpectedly however, within the SorT homodimer the protomers
136 are oriented in a head-to-tail orientation (Figure 1A), a subunit arrangement that has
137 not been previously observed in structures of sulfite-oxidizing enzymes. In keeping
138 with the nomenclature applied to other structurally-characterized SOEs, a
6 139 ‘dimerization’ domain typically defines the interface between the two monomers (7,
140 11), but the structure of the SorT dimer does not follow this paradigm.
141
142 Nevertheless, the fold of the SorT monomers is similar to those of other SOEs (7, 11,
143 21), comprising a central ‘SUOX-fold’ domain (26) that harbors the Mo active site
144 and a ‘dimerization’ domain (Figure 1A). The SorT active site has a square-
145 pyramidal geometry seen in all other SOE structures with a five coordinate
146 molybdenum atom and a single tricyclic pyranopterin cofactor (Figure 1A, Table 2)
147 (27).
148
149 Single electron reduction of SorT to its EPR active MoV form was achieved using a
150 combination of Ti(III)citrate and a suite of organic redox mediators. The MoV EPR
151 spectrum is similar to the so-called ‘high pH’ EPR signature of SOEs (Figure 2A,
152 Table 3, Appendix 1). An additional feature is the presence of superhyperfine
153 coupling between the unpaired electron on the Mo ion and two nearby I = ½ nuclei.
154 This implies that the equatorially coordinated O-donor is an aqua ligand at pH 8
155 (Figure 2B(ii)) or that a hydroxido ligand is hydrogen bonded with a water molecule
156 whose proximal H-atom is coupled with the electron spin on Mo (Figure 2B(i),
157 Appendix 1).
158
159 Within the SorT dimer, the subunit interface involves both the SUOX and the
160 ‘dimerization’ domains (Figure 1A), resulting in a buried surface area of ca. 1280 Å2
161 per monomer, which is approximately 9% of the solvent-accessible surface of each
162 monomer. The functional consequences and structural origins of these significant
163 differences among the quaternary structures of SOEs are at this point unknown, as all
7 164 of these enzymes are highly catalytically active, have similar active site structures and
165 no known kinetic cooperativity that would imply a functional role for the different
166 oligomeric assemblies (8, 10, 28).
167
168 In complex, SorT and SorU form a SorU/SorT2/SorU assembly that reveals a
169 pathway for electron transfer.
170 Despite the dynamic nature of the SorT/SorU interaction, it was possible to co-
171 crystallize SorT with SorU, resulting in the crystal structure of the SorT/SorU
172 complex, where a single SorT/SorU entity is present per asymmetric unit, and the
173 application of crystallographic 2-fold symmetry reveals a SorU/SorT2/SorU assembly
174 (Figure 3A, Table 1). Small angle X-ray scattering (SAXS) with a sample prepared
175 as a stoichiometric mixture of SorT and SorU (Figure 3B, Table 4, Appendix Figure
176 1) confirmed that the structure observed in the crystal is preserved in solution.
177
178 The central assembly within the SorU/SorT2/SorU complex is the SorT homodimer,
179 which is identical to the structure of the SorT homodimer alone (Figures 1A and 3A,
180 Table 1). The structure of the SorU protein, both within the SorT/SorU complex and
181 when crystallized in isolation (Figure 1B, Table 1) is predominantly α-helical with
182 three major α-helices arranged to form a bundle that frames the heme-binding site
183 (Figure 1B) (C47XXC50H, axial ligands: His 51 and Met 87).
184
185 In the SorT/SorU complex, the SorU protein docks within a pocket adjacent to the
186 SorT active site, with the heme cofactor located at the protein-protein interface
187 (Figures 3A and 4A). The shortest ‘edge-to-edge’ distance between the SorT Mo
188 atom and the propionate group from the SorU heme c cofactor is 8.2 Å, which is well
8 189 within the distance for fast electron transfer through the protein medium (31).
190 PATHWAY analysis (32) (Table 5) further indicates that the dominant electron
191 tunneling pathway from SorT to SorU proceeds from the Mo atom, via the
- 192 coordinating H2O/OH , to the guanidinium group of SorT residue Arg 78 and across
193 the protein-protein interface to the heme propionate group and to the pyrrole ring of
194 heme c to the heme iron (Figure 4B, Table 5).
195
196 Conformational change reminiscent of an ‘induced fit’ mechanism facilitates
197 docking and electron transfer between SorT and SorU.
198 Specific structural adaptations of the SorU protein take place when the SorT/SorU
199 assembly is formed (Figure 5B). A surface loop on SorU (residues 82-93) moves
200 away from the SorT/SorU complex interface, leading to a reorientation of the heme
201 ligand residue Met 87 so that a different Met 87 rotamer coordinates the iron in the
202 SorU structures within and outside of the complex (Figure 5B). This change in the
203 structure of SorU is required to allow a Mo-heme edge-to-edge distance of ‘closest
204 approach’ of ca. 8 Å within the SorT/SorU assembly. Without this adjustment (for
205 example, if the SorU residue 82-93 loop structure remained rigid) the closest
206 approach for the redox cofactors would be ca. 10 Å.
207
208 Changes in the conformation of axial heme ligands are known to alter the orbital
209 interactions between the iron atom and the ligand, which can change the redox
210 properties of the heme group (33). However, this does not appear to be the case here
211 as the redox potential of the SorU heme was determined by optical
212 spectroelectrochemistry to be +108(±10) and +111(±10) mV vs. NHE (pH 8.0),
213 respectively, in the presence and absence of SorT (Table 6, Figure 6B).
9 214
215 The MoVI/V and MoV/IV redox potentials of SorT (+110(±10) mV and -18(±10) mV vs.
216 NHE (pH 8)) were determined by an EPR-monitored redox titration where the initial
217 EPR-silent MoVI form is reduced to the EPR-active MoV state (Figure 2A), which
218 then gives way to the EPR-silent MoIV form at low potential, resulting in a bell-
219 shaped curve (Table 6, Figure 6A). The MoVI/V redox potential at pH 8 matches that
220 of the ferric/ferrous SorU couple.
221
222 The structural change in SorU indicates that an ‘induced fit’ mechanism is responsible
223 for the formation of a productive SorT/SorU electron transfer complex. This type of
224 mechanism has in the past been used to describe electron transfer complexes
225 (involving electron transfer flavoproteins or ferredoxin reductases) where the protein
226 partners include mobile domains, and where conformational change is necessary for
227 the creation of high affinity protein-protein interfaces (34, 35). However, although
228 facilitating redox interactions, these systems are distinct from the docking mechanism
229 seen for the SorT and SorU proteins which accompanies modifications to the structure
230 of SorU and allows the two redox centers to attain positions of closest approach for
231 fast electron transfer.
232
233 The SorT/SorU interface features extensive electrostatic interactions.
234 The SorT/SorU complex interface shows significant charge complementarity, with the
235 negative charge on SorU correlating with a concentration of positive charges at the
236 SorU binding site on SorT (Figures 3C, 3D). Unlike other known cytochromes c that
237 can act as electron acceptors to SOEs (12, 30, 36), the electrostatic surface of SorU
238 has an overall negative charge (Figures 3C, 3D). The positive charge on the SorT
10 239 surface therefore explains the low catalytic activity of SorT with horse heart
240 cytochrome c (7 U/mg, pI ~10), the natural electron acceptor for vertebrate SOEs,
241 compared to the high activity observed with SorU (212 U/mg; pI ~4) (5, 12, 17). In its
242 electrostatic nature, the interaction surface between SorT and SorU is unusual.
243 Structures of other cytochrome-containing electron transfer complexes (37-40) show
244 binding interfaces characterized by a ‘ring’ of electrostatic interactions that
245 encompass contact surfaces that are predominantly hydrophobic. In fact, the ‘steering’
246 of electron transfer partners by electrostatic interactions, accompanied by ‘tuning’ via
247 hydrophobic interactions is a dominant observation for protein-protein electron
248 transfer complexes (37-40).
249
250 An unusually large number of hydrogen bonds and salt bridges characterize the
251 SorT/SorU interface.
252 In addition to the electrostatic interactions that support the formation of the
253 SorT/SorU complex, there are six hydrogen bonds found at the SorT/SorU protein-
254 protein interface, as well as a salt bridge between the SorT active site residue Arg 78
255 and a propionate group of the SorU heme moiety (Table 7, Figure 7). This is an
256 unusually large number in comparison with structures of other cytochrome-containing
257 electron transfer complexes (37-40), which tend to have fewer hydrogen bonds and
258 lack salt bridges. In fact, direct hydrogen bonds between electron transfer proteins are
259 generally considered unfavorable for a transient interaction because of energetically
260 disadvantageous desolvation (41). Also significant is the observation that no
261 intermolecular interactions at the interface are mediated by water molecules (Figure
262 7) (37, 42). In fact, very little ordered water is observed in proximity to the interfacing
263 region of the SorT molecule (a total of 2 water molecules only and these are
11 264 hydrogen-bonded to the SorT molecule rather than mediating the SorT/SorU
265 interaction). However, the current analysis is limited by the moderate resolution of the
266 current structure (2.6 Å), which as a consequence, includes only ca. 0.15 modeled
267 water molecules per residue.
268
269 Compared with the subunit interface of the SorAB bacterial sulfite dehydrogenase,
270 which contains 30 hydrogen-bonds and 2 salt bridges (21), supporting a permanent,
271 heterodimeric complex of a heme c subunit (SorB) and a Mo cofactor containing
272 (SorA) catalytic subunit (21) (Table 7), the extent of the subunit interactions in the
273 SorT/SorU structure is modest. The difference between the permanent SorAB and the
274 transient SorT/SorU complex is also illustrated by calculations of the shape
275 complementarity and the buried surface areas between the protomers (43), with the
276 latter being about twice as large for the SorAB assembly than for SorT/SorU (Table
277 7). Interestingly, much of the additional contact area between molecules in the SorAB
278 structure derives from the SorB N-terminal structure (residues B501-B518, PDB
279 2BLF), which extends away from the core of the subunit, wraps around the SorA
280 ‘SUOX’ fold domain and contributes one salt-bridge and 6 hydrogen-bonding
281 interactions (Table 7) (21). This feature is absent from the SorU structure.
282
283 The dynamic SorT/SorU interaction observed in solution is reflected in the
284 crystal.
285 Despite the intricate assembly of interactions at the protein-protein interface, but in
286 agreement with the kinetics and thermodynamics of the SorT/SorU interaction in
287 solution, the SorT/SorU contact is dynamic, as illustrated by a temperature factor
288 analysis of the complex structure. Within the SorT/SorU complex, the SorU protein
12 289 shows a significantly increased average atomic temperature factor (reflecting
290 significant flexibility) relative to the structure of the SorT protomer (1.5 versus 0.9 Å2,
291 respectively; Table 7). Furthermore, the relative temperature factors per residue for
292 the SorU molecule increase with increasing distance from the SorT/SorU interface
293 (Figure 5A), indicating that the SorU molecule is dynamic relative to SorT within the
294 crystalline lattice. In contrast, the ‘static’ SorAB complex shows uniform, low
295 temperature factors for both redox subunits (Table 7, Figure 5A). This observation is
296 an exquisite illustration of ‘conformational sampling’ within the SorT/SorU electron
297 transfer complex, which results from the conformational flexibility of one protein
298 redox partner relative to the other and both facilitates electron exchange by accessing
299 the optimal orientations of each redox partner and promotes fast dissociation of the
300 complex following transfer (19, 44).
301
302 DISCUSSION
303 By describing the structure of the SorT/SorU complex in this work, we report the first
304 example of a structure of an SOE in complex with its external electron acceptor; all
305 previous structures of SOEs being of the enzymes and their internal heme domains or
306 subunits only (7, 11, 21). The structure of the SorT/SorU complex therefore allows
307 insights into electron transfer in what is thought to be a highly prevalent type of
308 bacterial SOE (12) and into protein-protein electron transfer in general. While the
309 complex shows dynamic adaptations similar to those demonstrated previously for
310 electron transfer complexes and has a dissociation constant of the right order of
311 magnitude, it also shows some features that have not been seen in electron transfer
312 complexes, namely an interface that is stabilized by a relatively large number of
313 hydrogen bonds and salt bridges, and an induced fit docking mechanism.
13 314
315 The structure of the protein-protein interface in the SorT/SorU structure is particularly
316 intriguing. The relative lack of bound water molecules, mirrors more the observations
317 made of permanent heterodimeric complexes than transient interactions (42). Previous
318 investigations into the factors that influence protein-protein docking for electron
319 transfer have shown that the strength of the protein-protein interaction correlates
320 linearly with the product of the total charges on the protein partners (45, 46). In this
321 way, the affinity between the SorT/SorU proteins (Kd = 13.5 ± 0.8 μM) correlates
-1 322 with the fast measured turnover rates (kcat of 140 ± 11 s ) and with the predominantly
323 electrostatic nature of the protein-protein interface.
324
325 The turnover number for SorT with SorU as the electron acceptor is also in the range
326 of values measured for the human sulfite oxidase (HSO) and CSO (25.0 ± 1.3 s-1 and
327 47.5 ± 1.9 s-1, respectively), but significantly slower than that observed for the
328 permanent SorAB complex (345 ± 11 s-1) (25, 28, 30). Importantly, in the structure of
329 SorAB, the docking site of the heme subunit (SorB) with the SorA is almost
330 identically positioned to that seen for SorT/SorU, with major differences existing only
331 in the number of hydrogen bonds and salt bridges in the protein-protein interface. For
332 the CSO and HSO enzymes, docking of the mobile heme b domain near the Mo active
333 site has been proposed to be similar to that seen for SorT/SorU (29). It should be
334 noted, however, that the docking events in CSO (and HSO) and between SorT/SorU
335 serve fundamentally different purposes: for CSO and HSO, domain docking enables
336 intramolecular electron transfer and involves a heme domain that is an intrinsic part of
337 the enzyme. This is a step that precedes interactions with the external electron
338 acceptor for these enzymes. In contrast, and despite the fact that it is occupying a
14 339 similar docking site to that predicted for CSO and HSO (29), SorU is the external
340 electron acceptor for SorT and the electron transfer is intermolecular.
341
342 The SorT/SorU complex described here thus represents an elegant compromise
343 between the requirements for fast and efficient electron transfer and reaction
344 specificity. It also illustrates new aspects for highly dynamic protein-protein
345 interactions: (i) A relatively large number of hydrogen bonds and salt bridges may be
346 required to form the initial stable protein complex, but this does not preclude a
347 dynamic protein – protein interaction; (ii) relatively subtle structural adjustments in
348 one redox partner (SorU) can facilitate electron transfer by ideally locating the redox
349 active cofactors in close proximity; (iii) the comparatively complex binding interface
350 in SorT/SorU can be counterbalanced by the conformational sampling of one protein
351 relative to the other, which enables the rapid dissolution of the complex following
352 electron exchange.
353
354 It remains to be seen whether these principles apply to other SOE – external electron
355 acceptor interactions. Future work should focus on investigating interaction interfaces
356 in currently little studied SOEs where new types of interactions may be present as for
357 many of these enzymes currently no external electron acceptor is known.
358
15 359 MATERIALS AND METHODS.
360 Protein overexpression, purification, data collection and structure solution.
361 Recombinant SorT and SorU proteins were overproduced and purified as previously
362 described (12), with minor modifications. SorT was crystallized by hanging drop
363 vapor diffusion with drops consisting of equal volumes (2 μL) of protein and
364 crystallization solution (0.1 M HEPES pH 7.5, 8% ethylene glycol, 0.1 M manganese
365 (II) chloride tetrahydrate and 17.5% PEG 10,000) at 20°C. Crystals were
366 cryoprotected in reservoir solution with 30% glycerol before flash cooling in liquid
367 nitrogen. Small (ca. 20 × 10 × 10 μ) crystals of SorU were grown in drops containing
368 equal volumes (2 μL) of protein and reservoir solution (1.8 M tri-sodium citrate, pH
369 5.5, 0.1 M glycine), which were harvested and flash-cooled in liquid nitrogen without
370 additional cryoprotection. Purified SorT (20 mM Tris pH 7.8, 2.5% glycerol) and
371 SorU (20 mM Tris pH 7.8, 150 mM NaCl) were mixed and incubated on ice at a
372 molar ratio of 2:1 (SorU:SorT; total protein concentration 8 mgmL-1) before
373 crystallization via hanging-drop vapor diffusion with a reservoir solution containing
374 0.2 M sodium formate, 0.1 M Bis-Tris propane pH 7.5 and 20% PEG 3350. Crystals
375 grew to a maximum size of ca. 150 x 100 x 20 μ in 4 days at 20°C and were flash
376 cooled in liquid nitrogen after brief soaking in mother liquor containing 30% glycerol.
377 All diffraction data were collected on an ADSC Quantum 315r detector at the
378 Australian Synchrotron on beamline MX2 at 100 K and were processed with
379 HKL2000 (47). Unit cell parameters and data collection statistics are presented in
380 Table 1.
381
382 The crystal structure of SorT was solved by molecular replacement using PHASER
383 (48) with a search model generated with CHAINSAW (49) from the SorA portion of
16 384 the SorAB crystal structure (29.0% sequence identity, Protein Data Bank entry 2BLF
385 (21)) as a template (50). The resulting model was refined by iterative cycles of
386 amplitude based twin refinement (using twin operators H, K, L and –H, -K, L with
387 estimated twin fractions of 0.495 and 0.505 respectively) within REFMAC (51),
388 interspersed with manual inspection and correction against calculated electron density
389 maps using COOT (52). The refinement of the model converged with residuals R =
390 0.208 and Rfree = 0.239 (Table 1). The structure of the SorT/SorU complex was
391 solved by molecular replacement using PHASER (48), with the refined SorT structure
392 as a search model. Initial rounds of refinement yielded a difference Fourier electron
393 density map, which clearly showed positive difference density for the location of one
394 molecule of SorU per asymmetric unit, which was manually built using COOT (52).
395 Refinement was carried out with REFMAC5 (51) and PHENIX (53) and converged
396 with residuals R = 0.211 and Rfree = 0.260 (Table 1). The refined SorU model, from
397 the SorT/SorU complex structure, was used as a search model to solve the SorU
398 structure by molecular replacement using PHASER (48). Refinement was carried out
399 with REFMAC5 and PHENIX (53) and converged with residuals R = 0.192 and Rfree
400 = 0.240. All structures were judged to have excellent geometry as determined by
401 MOLPROBITY (54)(Table 1).
402
403 Small Angle X-ray Scattering (SAXS).
404 SAXS analysis of the SorT/SorU complex was performed in a buffer of 20 mM Tris
405 pH 7.8, 2.5% v/v glycerol. Purified SorU and SorT were mixed and incubated on
406 ice at a molar ratio of 2:1 (SorU:SorT), generating two samples of total protein
407 concentrations 2.75 and 6.25 mgmL-1. SAXS data were measured as described
408 previously (55) with the data collection parameters listed in Table 4. Data were
17 409 reduced to I(q) vs q (q=4πsinθλ, where q=4sin2θ is the scattering angle) using the
410 program SAXSquant that includes corrections for sample absorbance, detector
411 sensitivity, and the slit geometry of the instrument. Intensities were placed on an
412 absolute scale using the known scattering from H2O. Protein scattering was obtained
413 by subtraction of the scattering from the matched solvents (20 mM Tris pH 7.8, 2.5%
414 v/v glycerol obtained from the flow-through after protein concentration by centrifugal
415 ultrafiltration). Molecular weight (Mr) estimates for the proteins were made using the
416 equation from Orthaber (62): Mr=NAI(0)CΔρM2 where NA is Avogadro’s number, C
417 is the protein concentration and ΔρM=Δρυ, where Δρ is the protein contrast and υ the
418 partial specific volume, both of which were determined using the program MULCh
419 (63).
420 The ATSAS program package (56) was used for data analysis and modeling, with the
421 specific programs used detailed in Table 4, along with the data ranges and results of
422 each of the calculations. Further detail on data interpretation and analysis for these
423 experiments is detailed in Appendix 2.
424
425 Electron Paramagnetic Resonance (EPR) spectroscopy
426 Continuous-wave X-band (ca. 9 GHz) (CW) electron paramagnetic resonance (EPR)
427 spectra were recorded with a Bruker Elexsys E580 CW/pulsed EPR spectrometer
428 fitted with a super high Q resonator; the microwave frequency and magnetic field
429 were calibrated with a Bruker microwave frequency counter and a Bruker ER 036TM
430 Teslameter, respectively. A microwave power of 20 mW was used and optimal
431 spectral resolution was obtained by keeping the modulation amplitude to a 1/10 of the
432 linewidth. A flow-through cryostat in conjunction with a Eurotherm (B-VT-2000)
18 433 variable temperature controller provided temperatures of 127-133 K at the sample
434 position in the cavity.
435
436 Bruker’s Xepr (version 2.6b.45) software was used to control the data acquisition
437 including, spectrometer tuning, signal averaging, temperature control and
438 visualization of the spectra. Computer simulation of the EPR spectra were performed
439 with the following spin Hamiltonian (Eq. 2)
2 440 H = βB⋅ g⋅S + S ⋅ A()95,97Mo ⋅ I − g β B ⋅ I + ()S ⋅ A()1H ⋅ I − g β B⋅ I (2) n n i=1 n n
441 using the XSophe-Sophe-XeprView (version 1.1.4) computer simulation software
442 suite (64, 65) on a personal computer, running the Mandriva Linux v2010.2 operating
443 system. Further detail on data interpretation and analysis for these experiments is
444 detailed in Appendix 1.
445
446 EPR-monitored redox potentiometry
447 The MoIV/V and MoV/VI redox potentials of SorT were determined by an EPR
448 monitored redox titration carried out in a Belle Technology anaerobic box. The
449 protein solution (1.5 mL, 40-90 μM in Tris-HCl, pH 8.0 and 10% glycerol) also
450 contained the following redox mediators at concentrations of ~50 µM: diaminodurol
451 (2,3,5,6-tetramethylphenylene-1,4-diamine, Em,7 +276 mV),
452 dichlorophenolindophenol (Em,7 +217 mV), 2,6-dimethylbenzoquinone (Em,7 +180
453 mV), phenazine methosulfate (Em,7 +80 mV), 2,5-dihydroxybenzoquinone (Em,7 –60
454 mV), indigo trisulfonate (Em,7 –90 mV), 2-hydroxy-1,4-naphthoquinone (Em,7 -152
455 mV) and anthraquinone 2-sulfonate (Em,7 -230 mV). The reductant was Ti(III) citrate
456 and the oxidant was NaS2O8 (both ~100 mM). After addition of titrant and
457 equilibration (15-30 min), the equilibrium potential was measured with a combination
19 458 Pt wire/AgAgCl redox electrode attached to a Hanna 8417 meter calibrated against
459 the quinhydrone redox couple (Eo′ (pH 7) = +284 mV vs NHE). A 100 μL aliquot of
460 protein was withdrawn and transferred to an EPR tube (in the anaerobic box) which
461 was then sealed and then carefully frozen in liquid nitrogen (outside the box).
462 Potentials for all experiments were measured with a combination Pt wire-Ag/AgCl
463 electrode attached to a Hanna 8417 meter. The intensity of the MoV signal (I) was
464 recorded as a function of measured potential (E), Figure 6.
465
466 Optical Spectroelectrochemistry
467 Spectroelectrochemistry of SorU in isolation and the SorT:SorU complex was
468 performed with a Bioanalytical Systems BAS100B/W potentiostat connected to a
469 Bioanalytical Systems thin layer spectroelectrochemical cell (0.5 or 1 mm pathlength)
470 bearing a transparent Au mini-grid working electrode, a Pt wire counter and Ag/AgCl
471 reference electrode. Redox mediators (all Fe complexes) used in the experiment were
472 employed at concentrations of 50 μM (Scheme (2)).
473 (2)
474 None exhibit any significant absorption in the spectral range of interest at micromolar
475 concentrations. The total solution volume was ca. 700 μL. The buffer was 20 mM
476 Tris (pH 8) containing 200 mM NaCl as supporting electrolyte. The SorU
477 concentration was ca. 50 μM while experiments on the SorU:SorT complex used
478 approximately equal concentrations of both proteins (50 μM). Spectra were acquired
479 within a Belle Technology anaerobic box with an Ocean Optics USB2000 fibre optic
480 spectrophotometer. Initially the cell potential was poised at ca. -100 mV and the
20 481 system was allowed to equilibrate until no further spectral changes were apparent
482 (fully reduced SorU). The potentials were then increased in 50 mV increments and the
483 spectrum was measured when no further changes were seen (5-10 min). When the
484 protein was fully oxidized the potential was scanned in the reverse direction in 50 mV
485 intervals to establish reversibility. Data were fitted using the program ReactLab
486 Redox (58).
487
488 Isothermal Titration Calorimetry (ITC)
489 Affinity measurements were conducted using a Microcal ITC200 system (GE
490 Healthcare) at 25°C using SorT and SorU in buffer (25 mM HEPES pH 7.5, 150 mM
491 NaCl and 2.5 % glycerol) at final concentrations of 300 μM and 30 μM, respectively.
492 SorT at a concentration of 300 μM was titrated with eighteen injections (2.0 μl each)
493 of SorU. All affinity measurements were performed in triplicate and fitted using a
494 single site mode. Protein concentrations were estimated using Bicinchoninic acid
495 (BCA) protein assay kit (ThermoScientific).
496
497 Enzyme kinetics
498 Sulfite dehydrogenase enzyme assays were carried out as described previously (12, 17,
499 36). The reduced – oxidized extinction coefficient for SorU at 550 nm was 17.486
500 mM-1 cm-1 as determined by spectroelectrochemistry. Data fitting was carried out
501 using Sigmaplot 12 (Systat).
502
21 503 ACKNOWLEDGEMENTS.
504 This work was supported by a La Trobe Institute for Molecular Science (LIMS)
505 Senior Research Fellowship to MJM an Australian Research Council grant and
506 Fellowship (DP0878525) to UK and an NHMRC CJ Martin Postdoctoral Research
507 Fellowship to APM. PVB also acknowledges financial support from the Australian
508 Research Council (DP1211465). Aspects of this research were undertaken on the
509 Macromolecular Crystallography beamline at the Australian Synchrotron (Victoria,
510 Australia) and we thank the beamline staff for their enthusiastic and professional
511 support. A/Prof Matthew Perugini (La Trobe University) is thanked for very helpful
512 discussions. Assistance with EPR spectroscopy from Dr Jeff Harmer and Dr Chris
513 Noble (Centre for Advanced Imaging, University of Queensland) is also gratefully
514 acknowledged.
515
516
517
518
519
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32 739 FIGURE TITLES AND LEGENDS.
740 Figure 1. The crystal structures of the SorT and SorU proteins in isolation. Panel
741 A: The structure of the SorT homodimer. Molecule A in blue/yellow; molecule B in
742 gray (with transparent surface). For molecule A, the ‘SUOX-fold domain’ and
743 ‘dimerization domain’ are represented in blue and yellow, respectively. The
744 molybdopterin cofactor is shown as sticks within the SUOX-fold domain. The
745 corresponding domains of the opposing protomer (shown in molecular surface
746 representation), which constitute the dimer interface are coloured to highlight the
747 ‘head to tail’ dimer arrangement. INSET: a closer view of the molybdenum binding-
748 site: the molybdenum atom (green sphere) is coordinated by two dithioline ligands
749 from the molybdopterin (yellow spheres), residue Cys 127, an axial oxo ligand and an
750 equatorial hydroxo or water ligand (red spheres); Panel B: The structure of SorU. The
751 main three helices are labeled and the heme cofactor is shown in red. INSET: the
752 heme binding site with the heme cofactor, coordinating residues, covalent links to Cys
753 50 and 57 and hydrophobic residues lining binding site: Phe 39, Val 62, Val 76, Val
754 80, Val 101 highlighted.
755
756 Figure 2. EPR analysis of the SorT protein. Panel A. X-band EPR spectra of the
757 Mo(V) center in SorT. (a) First and (b) second derivative EPR spectra of SorT at +50
758 mV vs NHE in Tris-HCl pH 8.0, ν= 9.43462 GHz, T = 136.3 K. (c) Computer
759 simulation of the second derivative spectrum with the spin Hamiltonian parameters
760 listed in Table 3; (d,e) Expansion of spectra (b) and (c), respectively. Panel B.
761 Schematic structures of the (a) high and (b) low pH forms of sulfite oxidase.
762
33 763 Figure 3. The structure of the SorT/SorU electron transfer complex. Panel A.
764 The asymmetric unit from the crystal structure of the SorT/SorU complex contains the
765 functional electron transfer complex. The SorU/SorT2/SorU complex is revealed by
766 the application of crystallographic symmetry operators. The positions of the redox
767 active molybdenum (SorT) and heme c (SorU) cofactors are indicated. Panel B. Two
768 views of an overlay of the SorU/SorT2/SorU crystal structure with the averaged and
769 filtered dummy atom model from 10 ab initio reconstructions as revealed by SAXS
770 analyses. Panel C. ‘Open-book unfolding’ of SorT/SorU complex (SorT is shown in
771 blue, SorU in red) indicating the ‘footprint’ of interfacing residues from each protein.
772 Panel D. The same view as Panel C, showing the charge complementarity of the
773 SorT/SorU interface (areas of positive charge in blue, negative charge in red and
774 neutral in white).
775
776 Figure 4. Orientation of the redox cofactors in the crystal structure of the
777 SorT/SorU electron transfer complex. Panel A. Electron density map in the region
778 of the SorT/SorU interface. The SorT molecule is represented in blue and the SorU
779 molecule in red. The 2Fo-Fc electron density map is shown as a blue net and the
780 redox cofactors (molybdenum and heme) are colored according to the representation
781 in Panel B. Panel B. Pathway for electron transfer (57).
782
783 Figure 5. Comparisons of (A) the SorT/SorU and SorAB structures and (B) the
784 structures of SorT and SorU within and outside of the electron transfer complex.
785 Panel A. Structures of the SorT/SorU (left) and SorAB (right) complexes, where the
786 Cα traces of the heme-containing protomers are colored according to temperature
787 factor. Panel B. Superposition of the SorT and SorU structures within and outside of
34 788 the electron transfer complex, highlighting conformational changes that were
789 observed to accompany complex formation. The crystal structures of SorT and SorU
790 within the SorT/SorU complex are shown in gray, and the superposed structures of
791 SorT and SorU determined alone are shown in blue and red respectively.
792
793 Figure 6. Redox analyses of the SorT protein and the SorT/SorU complex. Panel
V 794 A. Plot of EPR intensity (Ip) at 343 mT (from Mo form of SorT) as a function of
795 solution redox potential (E mV vs NHE). The solid line is a fit to the equation
I p = VI/V 796 IE() −− using the potentials E1 = Mo = +110(±10) 110++()/59(EE12 10 E E )/59
V/IV 797 mV and E2 Mo -18(±10) mV vs NHE). Panel B. Electronic spectra of ferric and
798 ferrous SorU obtained from spectroelectrochemistry. Inset: plot of absorbance at 550
799 nm (ferrous α-band) and 406 nm (ferric Soret band) as a function of applied potential.
800 The solid lines are theoretical curves based on the equation
(10εε(')/59EE− + ) 801 Abs= ox red c where the extinction coefficients refer to the 110+ (')/59EE− tot
802 oxidized and reduced forms of the protein and Abs is the absorbance at this same
803 wavelength. ctot is the total protein concentration. The redox potential (E' = +111 mV
804 vs NHE) was obtained by global analysis of all potential dependent spectra across all
805 wavelengths with the program ReactLab Redox (58).
806
807 Figure 7. The bonding network at the interface of SorT/SorU. Panel A: An open-
808 book representation depicts residues involved in forming stable bonds at the interface
809 between SorT and SorU as corresponding color patches mapped onto the molecular
810 surface. Panel B: Stereoview of the interface between SorT and SorU. Bonding
35 811 residues are shown as sticks with bonds shown as dashes between atoms. SorT is
812 shown in light grey and SorU is shown in magenta.
813
814 Appendix Figure 1. SAXS Data and Interpretation. Panel A. Guinier plot of the
815 desmeared I(q) versus q; Panel B. log:log plot of the measured (slit smeared) I(q)
816 versus q with the P(r)- model I(q) (black line) and smeared P(r)-model I(q) (red line)
817 fits; Panel C. P(r) versus r for the P(r) model in (B), dmax is 110 Å; Panel D.
818 superposition of the desmeared I(q) versus q with that calculated from the crystal
819 structure of the SorT/SorU2/SorT complex (red line) and the SorT dimer (green line).
820
821
36 Table 1. Data collection and refinement statistics
SorT SorU SorT/SorU complex
Data collection
Space Group P21 F222 P21212
Cell dimensions
a, b, c (Å) 96.0, 92.2, 109.4 70.9, 129.2, 197.0 109.6, 95.8, 49.9
α, β, γ (°) 90, 89.7, 90 90, 90, 90 90, 90, 90
X-ray source AUS MX2 AUS MX2 AUS MX2
λ (Å) 0.950 0.954 0.954
Detector ADSC Quantum 315r ADSC Quantum 315r ADSC Quantum 315r
Resolution range (Å) 50-2.4 (2.43-2.35)a 50-2.2 (2.28-2.20) 50-2.5 (2.50-2.59)
Observed reflections 240521 96340 64273
Unique reflections 77971 23453 18883
Completeness (%) 98.4 (99.4) 99.9 (100) 99.3 (99.6)
Multiplicity 3.1 (3.1) 4.1 (4.1) 3.4 (3.4)
6.7 (2.1) 8.9 (1.6) 8.8 (1.7)
b Rmerge (%) 15.9 (66.7) 13.3 (76.6) 13.9 (76.6)
Refinement
Reflections in working 74024 22113 17781 set
Reflections in test set 3927 1201 960
Protomers per ASU 4 4 1
Total atoms (non-H) 11378 2941 3422
Protein atoms 10890 2542 3290
37 Metal atoms 4 4 2
Water atoms 376 227 63
Other atoms 108 168 67
c Rwork (%) 20.8 (31.7) 19.2 (30.8) 21.1 (30.2)
d Rfree (%) 23.9 (34.7) 24.0 (34.3) 26.0 (36.5)
Rmsd bond lengths (Å) 0.008 0.006 0.012
Rmsd bond angles (deg) 1.08 0.91 1.41
(Å2)e 32.5 20.6 38.0
Cruickshank's DPI 0.07 0.23 0.49
PDB ID 4PW3 4PWA 4PW9
aValues in parenthesis are for highest-resolution shell
b Rmerge = ∑hkl ∑i⏐Ii (hkl) - I(hkl)⏐/∑hkl ∑i Ii (hkl)
c Rwork = Σh ⏐Fobs – Fcalc⏐/ΣhFobs
d Calculated as for Rwork using 10% of the diffraction data that had
been excluded from the refinement
eAs calculated by BAVERAGE (59, 60)
822
823
38 824 Table 2. Mo coordination geometry in the active site of SorT.
Bond Distance (Å)
Mo-S1 (pterin) 2.4
Mo-S2 (pterin) 2.4
Mo-S (Cys 127) 2.3
Mo=O 1.7
Mo-OH/H2O 1.9
825
826
39 827 Table 3. Spin Hamiltonian parameters for the Mo(V) center of SorT and various
828 low and high pH forms of human, avian, plant and bacterial sulfite oxidases.
829
Species Parameter X Y Z βo Ref
SorT g 1.94930 1.95997 1.98632 -
A(95Mo)b 20.5 36.0 53.9 26
A(1H)b,c 3.5 4.0 4.8 0
SorAc g 1.9541 1.9661 1.9914 -d 56
Human SO g Low pH 1.9646 1.9723 2.0023 - 52
A(1H)b 11.47 7.10 7.71 - 52
Chicken SO g (Low pH) 1.9658 1.9720 2.0037 - 51
A(1H)b 11.93 7.37 7.95 - 51
g (High pH) 1.9531 1.9641 1.9872 51
A. Thaliana g (Low pH) 1.963 1.974 2.005 - 57
SO
A(1H)b 11.9 9.2 10.3 -- 57
g (High pH) 1.956 1.964 1.989 - 57
830
831 a Non coincident angle between g and A (rotation about x axis). bUnits 10-4 cm-1. c
832 Two magnetically equivalent protons (I=1/2) were included in the computer simulated
c 95 833 spectra. Mo hyperfine couplings were unresolved and the shoulders on gz were
834 incorrectly attributed to 95Mo hyperfine resonances. dEuler angles were not
835 determined.
836
40 837 Table 4. Data collection and processing parameters for analysis of the SorT/SorU
838 complex in solution by Small Angle X-ray Scattering (SAXS).
839
Data collection parameters
Instrument SAXSess (Anton Paar)
Beam geometry 10 mm slit
AH, LH (Å-1), GNOM beam geometry definition 0.28, 0.12
q-range measured (Å-1) 0.01-0.400
Exposure time (min) 60 (4 x 15)
-1 SorT2SorU2 concentration range (mg mL ) 2.75-5.5
Temperature (ºC) 10
Structural parameters*
-1 Rg (Å), I(0) (cm ) 30.8 ± 0.4, 0.223 ± 0.002
from Guinier (desmeared data) q*Rg < 1.3
-1 Rg (Å), I(0) (cm ) 32.0 ± 0.3, 0.235 ± 0.002
from P(r) (q-range 0.01 – 0.25 Å-1)
dmax (Å) from P(r) 110
Molecular mass determination*
Molecular mass Mr from Guinier I(0) (ratio with 108741 (0.984)
expected)
Molecular mass Mr from P(r) I(0) (ratio with expected) 114593 (1.037)
SorT2SorU2 parameters calculated from sequence and chemical composition
Molecular volume (Å3) 134385
Molecular weight Mr (Da) 110556
41 Partial specific volume (cm3 g-1) 0.732
Contrast (X-rays) (Δρ x 1010 cm-2) 2.895
Modeling results and validation
Crystal structure Rg, dmax (Å)
SorT/SorU2/SorT 31.3, 108
SorT 27.9, 99
Crystal structure compare to desmeared I(q) (χ-value)
-1 SorT/SorU2/SorT (q-range 0.01 – 0.15 Å ) 1.7
SorT (q-range 0.01 – 0.15 Å-1) 2.3
Results from 10 ab initio shape restorations. P1 symmetry:
Average molecular volume (Å3) 140800
Normalised spatial distribution (NSD) and NSD 0.508 (0.008) variation
χ value for fit to desmeared data 1.8
Software employed
Calculation of expected Mr, Δρ and υ values MULCh
Primary data reduction, I(q) vs q SAXSQuant 1D
Desmearing SAXSQuant
Guinier analysis PRIMUS
P(r) analysis GNOM
Model I(q) from crystal coordinates CRYSOL ab initio shape restorations DAMMIN
42 3D graphics representations PYMOL
840 *Reported for 2.75 mg ml-1 measurement.
841
43 842 Table 5. Electron transfer parameters between SorT (Mo) and SorU (Fe) (32)
Distance (Mo-Fe, Å) 16.5 Å
Atomic packing density (ρ) 0.97
Average decay exponential (β) 0.97
-4 Electronic coupling (HDA) 3.4 x 10
Maximum ET rate (s-1) 1.2 x 107
843
844
44 845 Table 6. Redox potential values for SorT and SorU at pH8a.
Protein Couple Eº (mV vs NHE)
SorT MoVI/V +110(±10)
MoV/IV -18(±10)
SorU FeIII/II +108 (±10)
SorU FeIII/II +111 (±10)
(in the presence of SorT)
846 aRedox potentials of SorT were determined by redox potentiometry, and SorU redox
847 potentials by optical spectroelectrochemistry.
848
45 849
850 Table 7. Comparison of the protein-protein interfaces in the SorT/SorU and
851 SorAB structures.
852
Parameter SorT/SorUa SorABb
SorT SorU SorA SorB
Average relative B factorc 0.9 1.5 1.0 1.1
(Å2)
Buried surface area (Å2)d 644 696 1254 1380
Interfacing residuesd 31 21 46 33
Hydrogen-bonds 6 30e
Salt-bridges 1 2e
Shape complementarity 0.63 0.77
statisticf
853 a This work
854 b PDB code 2BLF (21)
855 c Calculated as the average for the protomer of interest divided by the average for the
856 entire complex structure.
857 d (61)
858 e Taken from (21)
859 f (43)
860
861
862
863
46 864 APPENDIX 1
865 SUPPLEMENTAL MATERIAL ON THE INTERPRETATION OF EPR DATA
866 The optimum potential to measure the continuous wave (CW) EPR spectrum of the
867 Mo(V) center in SorT was found to be +50 mV from the redox potentiometry
868 experiments (Figure 6A). The CW EPR spectrum at 0 mV (Figure 2A(a)) arises
869 from a single rhombically distorted Mo(V) center.
870
871 Naturally abundant Mo consists of a mixture of isotopes (95,97Mo, I=5/2, 25.5 %
872 abundance; 92,94,96,98,100Mo, I=0, 74.5 % abundance) and consequently the spectrum
873 consists of three I=0 resonances corresponding to the principal directions of the g-
874 matrix and six (2I+1) satellite resonances, each with an intensity of ~4%. Increased
875 spectral resolution was obtained by numerically differentiating the spectrum and
876 carefully Fourier filtering (Hamming function) the spectrum to remove the high
877 frequency noise without distorting the spectrum (Figure 2A(b)). Interestingly, a close
878 examination of the gz resonance around 339.35 mT reveals a triplet (Figure 2A) (64,
879 65) in an approximate ratio of 1:2:1. Computer simulation of the CW EPR spectrum
880 with a monoclinic (Cs symmetry) spin Hamiltonian (Eq. (2)) incorporating two
881 magnetically equivalent I=1/2 nuclei and the spin Hamiltonian parameters listed in
882 Table 3 produces the spectrum shown in Figure 2A(c).
883
884 Previously we have shown in a study of model Mo(V) complexes that the non
885 coincident angle, β (rotation of Ay,z from gy,z about x) can be accurately determined
886 utilizing multifrequency CW EPR in conjunction with computer simulation studies
887 (66, 67). Herein, the increased spectral resolution in the second derivative spectrum
888 and the narrow line widths enable an accurate determination of the non coincident
47 889 angle β without resorting to the use of multiple microwave frequencies. Computer
890 simulation of the second derivative spectrum showed that the 95,97Mo hyperfine
891 resonant field positions along the ‘z’ and ‘y’ directions were highly sensitive to the
892 magnitude of these hyperfine couplings and the non coincident angle β. The excellent
893 agreement between the simulated and experimental second derivative spectra (Figure
894 2A(b)(c)) gives confidence in the values of the spin Hamiltonian parameters. We have
895 also shown through a systematic density functional theory study that the non
896 coincident angle β can be correlated to the pterin fold angle (68) and for β=26o, the
897 predicted pterin fold angle would be 5.6o, which is in good agreement with that
898 determined (1.9o±0.2o) from the X-ray crystal structure of SorT.
899
900 CW and pulsed EPR spectra of the Mo(V) center in human, avian, plant and bacterial
901 sulfite have been extensively studied (Table 3) (36, 69-75). The Mo(V) CW EPR
902 spectra of SO from humans, birds and plants are pH dependent. The CW EPR spectra
903 arise from a Mo(V) center with rhombic or lower symmetry and at low pH the
904 resonances are split into a doublet arising from a strongly coupled proton (Table 3).
905 CW and pulsed EPR spectra in conjunction with isotope enrichment (17O, 2H) studies
906 have identified the origin of the proton as an equatorial hydroxo ligand. Loss of the
907 proton superhyperfine coupling at high pH in the CW EPR spectrum, was shown
908 through orientation selective multifrequency electron spin echo envelope modulation
909 (ESEEM) studies to be a rotation of the hydroxo moiety out of the xy plane (rather
910 than deprotonation), thereby reducing the unpaired electron spin density on the 1H
911 nucleus of the hydroxo ligand (69, 73, 76). 17O ESEEM studies also revealed two
912 other weakly coupled oxygen ligands, the axially coordinated Mo=O moiety and a
48 913 weakly coupled OH- moiety hydrogen bonded to the equatorial hydroxyo ligand (73,
914 76).
915
916 In contrast, the Mo(V) EPR spectra of SOEs from bacteria (SorA (36) and SorT) are
917 pH independent. A comparison of the gz resonances from SorA (36) and SorT
918 (Figure 2) shows their lineshapes to be very similar, both exhibiting shoulders.
919 Herein we have clearly shown through computer simulation of the Mo(V) CW EPR
920 spectrum that the shoulders do not arise from 95,97Mo hyperfine coupling (Figure 2,
921 Table 3), but arise from two weakly coupled 1H nuclei. While the computer
922 simulation assumed that the two protons were magnetically equivalent, a slight
923 magnetic inequivalence of the two 1H superhhyperfine couplings cannot be ruled out.
924 The origin of the 1H superhyperfine couplings is likely to be either (i) an equatorial
925 aqua ligand with the protons lying outside of the equatorial plane, thereby reducing
926 the overlap with Mo based dxy orbital containing the unpaired electron or (ii) an
927 equatorial hydroxo ligand which is hydrogen bonded to another hydroxyl moiety
928 (Figure 2B) where both protons lie outside of the equatorial plane. The proton
929 superhyperfine couplings are similar to those found from pulsed EPR studies of SorA
930 (69, 77).
931
932
933
934
935
936
937
49 938 APPENDIX 2
939 SUPPLEMENTAL MATERIAL FOR THE INTERPRETATION OF SAXS
940 DATA
941 The scattering data from the sample prepared as a stoichiometric mixture of SorT and
942 SorU is presented in Figure 3B and Figure 3-figure supplement 1, while Table 4
943 summarizes the structural parameters derived from those data. Significantly the Mr
944 value determined from I(0) is in excellent agreement with that expected for a 2:2
945 complex (within 2-4% for calculations based on Guinier or P(r)derived I(0)). Further,
946 the scattering data fit the crystal structure of the SorT/SorU complex reasonably well,
947 yielding a χ value of 1.7 for the overall fit and good agreement with the crystal
948 structure Rg value. This agreement is not expected to be perfect as the crystal structure
949 is missing a total of 56 residues: 33 from the N-terminus of the SorT, 2 from the N-
950 terminus of SorU and 21 from the C-terminus of SorU; accounting for 12% of the
951 total molecular mass. By comparison, a significantly worse χ value of 2.3 is obtained
952 by fitting only the SorT dimer, which also predicts a significantly smaller Rg value
953 than was observed (28 Å compared to 31 Å).
954
955 Dummy atom reconstructions yielded shapes that had the expected molecular volume
956 and in projection had the expected shape for the SorU/SorT2/SorU assembly, though
957 the shapes were consistently a somewhat flatter disk-like structure than that observed
958 in the crystal structure (Figure 3B). This result is not unexpected as this class of
959 structure (flattened anisotropic particles) is known to present some difficulty on ab
960 initio shape reconstructions (56).
50