1. Structural Biology and Molecular Biophysics
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Structural basis of interprotein electron transfer in bacterial sulfite oxidation

  1. Aaron P McGrath
  2. Elise L Laming
  3. G Patricia Casas Garcia
  4. Marc Kvansakul
  5. J Mitchell Guss
  6. Jill Trewhella
  7. Benoit Calmes
  8. Paul V Bernhardt
  9. Graeme R Hanson
  10. Ulrike Kappler  Is a corresponding author
  11. Megan J Maher  Is a corresponding author
  1. Centenary Institute, Australia
  2. University of Sydney, Australia
  3. La Trobe Institute for Molecular Science, La Trobe University, Australia
  4. The University of Queensland, Australia
  5. University of Queensland, Australia
Research Article
Cite this article as: eLife 2015;4:e09066 doi: 10.7554/eLife.09066
9 figures, 7 tables and 3 data sets

Figures

The crystal structures of the SorT and SorU proteins in isolation.

(A) The structure of the SorT homodimer. Molecule A in blue/yellow; molecule B in gray (with transparent surface). For molecule A, the ‘SUOX-fold domain’ and ‘dimerization domain’ are represented in blue and yellow, respectively. The molybdopterin cofactor is shown as sticks within the SUOX-fold domain. The corresponding domains of the opposing protomer (shown in molecular surface representation), which constitute the dimer interface are colored to highlight the ‘head-to-tail’ dimer arrangement. INSET: a closer view of the molybdenum binding-site: the molybdenum atom (green sphere) is coordinated by two dithioline ligands from the molybdopterin (yellow spheres), residue Cys 127, an axial oxo ligand and an equatorial hydroxo or water ligand (red spheres). (B) The structure of SorU. The main three helices are labeled and the heme cofactor is shown in red. INSET: the heme binding site with the heme cofactor, coordinating residues, covalent links to Cys 50 and 57 and hydrophobic residues lining binding site: Phe 39, Val 62, Val 76, Val 80, Val 101 highlighted.

https://doi.org/10.7554/eLife.09066.003
EPR analysis of the SorT protein.

(A) X-band EPR spectra of the Mo(V) center in SorT. (a) First and (b) second derivative EPR spectra of SorT at 0 mV vs NHE in tricine pH 8.0, υ= 9.43462 GHz, T = 136.3 K. (c) Computer simulation of the second derivative spectrum with the spin Hamiltonian parameters listed in Table 3; (d,e) Expansion of spectra (b) and (c), respectively. (B) Schematic structures of the (i) high and (ii) low pH forms of sulfite oxidase.

https://doi.org/10.7554/eLife.09066.006
The structure of the SorT/SorU electron transfer complex.

(A) The asymmetric unit from the crystal structure of the SorT/SorU complex contains the functional electron transfer complex. The SorU/SorT2/SorU complex is revealed by the application of crystallographic symmetry operators. The positions of the redox active molybdenum (SorT) and heme c (SorU) cofactors are indicated. (B) Two views of an overlay of the SorU/SorT2/SorU crystal structure with the averaged and filtered dummy atom model from 10 ab initio reconstructions as revealed by SAXS analyses. (C) ‘Open-book unfolding’ of SorT/SorU complex (SorT is shown in blue, SorU in red) indicating the ‘footprint’ of interfacing residues from each protein. (D) The same view as Panel C, showing the charge complementarity of the SorT/SorU interface (areas of positive charge in blue, negative charge in red and neutral in white).

https://doi.org/10.7554/eLife.09066.008
Orientation of the redox cofactors in the crystal structure of the SorT/SorU electron transfer complex.

(A) Electron density map in the region of the SorT/SorU interface. The SorT molecule is represented in blue and the SorU molecule in red. The 2Fo-Fc electron density map (contoured at 1σ) is shown as a blue net and the redox cofactors (molybdenum and heme) are colored according to the representation in Panel B. (B) Pathway for electron transfer (Beratan et al., 1992).

https://doi.org/10.7554/eLife.09066.010
Comparisons of (A) the SorT/SorU and SorAB structures and (B) the structures of SorT and SorU within and outside of the electron transfer complex.

(A) Structures of the SorT/SorU (left) and SorAB (right) complexes, where the Cα traces of the heme-containing protomers are colored according to temperature factor. (B) Superposition of the SorT and SorU structures within and outside of the electron transfer complex, highlighting conformational changes that were observed to accompany complex formation. The crystal structures of SorT and SorU within the SorT/SorU complex are shown in gray, and the superposed structures of SorT and SorU determined alone are shown in blue and red respectively.

https://doi.org/10.7554/eLife.09066.012
Redox analyses of the SorT protein and the SorT/SorU complex.

(A) Plot of EPR intensity (Ip) at 343 mT (from MoV form of SorT) as a function of solution redox potential (E mV vs NHE). The solid line is a fit to the equation I(E) = Ip1 =10(E-E1)/59 + 10(E2-E)/59 using the potentials E1 = MoVI/V = +110(±10) mV and E2 MoV/IV-18(±10) mV vs NHE). (B) Electronic spectra of ferric and ferrous SorU obtained from spectroelectrochemistry. Inset: plot of absorbance at 550 nm (ferrous α-band) and 406 nm (ferric Soret band) as a function of applied potential. The solid lines are theoretical curves based on the equation Abs = (εox10(E-E')/59) +εred)1 + 10(E-E')/59Ctot where the extinction coefficients refer to the oxidized and reduced forms of the protein and Abs is the absorbance at this same wavelength. ctot is the total protein concentration. The redox potential (E' = +111 mV vs NHE) was obtained by global analysis of all potential dependent spectra across all wavelengths with the program ReactLab Redox (Maeder and King).

https://doi.org/10.7554/eLife.09066.013
The bonding network at the interface of SorT/SorU.

(A) An open-book representation depicts residues involved in forming stable bonds at the interface between SorT and SorU as corresponding color patches mapped onto the molecular surface. (B) Stereoview of the interface between SorT and SorU. Bonding residues are shown as sticks with bonds shown as dashes between atoms. SorT is shown in light grey and SorU is shown in magenta.

https://doi.org/10.7554/eLife.09066.016
Redox mediators employed in optical spectroelectrochemistry experiments.
https://doi.org/10.7554/eLife.09066.017
Appendix 2-figure 1
SAXS Data and Interpretation.

(A) Guinier plot of the desmeared I(q) versus q; (B) log:log plot of the measured (slit smeared) I(q) versus q with the P(r)- model I(q) (black line) and smeared P(r)-model I(q) (red line) fits; (C) P(r) versus r for the P(r) model in (B), dmax is 110 Å; (D) superposition of the desmeared I(q) versus q with that calculated from the crystal structure of the SorT/SorU2/SorT complex (red line) and the SorT dimer (green line).

https://doi.org/10.7554/eLife.09066.018

Tables

Table 1

Data collection and refinement statistics.

https://doi.org/10.7554/eLife.09066.004
SorTSorUSorT/SorU complex
Data collection
Space GroupP21F222P21212
Cell dimensions
a, b, c (Å)
96.0, 92.2, 109.470.9, 129.2, 197.0109.6, 95.8, 49.9
α, β, γ (°)
90, 89.7, 9090, 90, 9090, 90, 90
X-ray sourceAUS MX2AUS MX2AUS MX2
λ (Å)0.9500.9540.954
DetectorADSC Quantum 315rADSC Quantum 315rADSC Quantum 315r
Resolution range (Å)50-2.4 (2.43-2.35)a50-2.2 (2.28-2.20)50-2.5 (2.50-2.59)
Observed reflections2405219634064273
Unique reflections779712345318883
Completeness (%)98.4 (99.4)99.9 (100)99.3 (99.6)
Multiplicity3.1 (3.1)4.1 (4.1)3.4 (3.4)
<I/σ(I)>6.7 (2.1)8.9 (1.6)8.8 (1.7)
Rmerge (%)b15.9 (66.7)13.3 (76.6)13.9 (76.6)
Refinement
Reflections in working set740242211317781
Reflections in test set39271201960
Protomers per ASU441
Total atoms (non-H)1137829413422
Protein atoms1089025423290
Metal atoms442
Water atoms37622763
Other atoms10816867
Rwork (%)c20.8 (31.7)19.2 (30.8)21.1 (30.2)
Rfree (%)d23.9 (34.7)24.0 (34.3)26.0 (36.5)
Rmsd bond lengths (Å)0.0080.0060.012
Rmsd bond angles (deg)1.080.911.41
<B> (Å2)e32.520.638.0
Cruickshank's DPI0.070.230.49
PDB ID4PW34PWA4PW9
  1. aValues in parenthesis are for highest-resolution shell

  2. b Rmerge = ∑hkl| Ii (hkl) - <I(hkl)> |/∑hkli Ii (hkl)

  3. c Rwork = ∑h | Fobs – Fcalc |/∑hFobs

  4. d Calculated as for Rwork using 10% of the diffraction data that had been excluded from the refinement

  5. eAs calculated by BAVERAGE (Winn et al., 2011)

Table 2

Mo coordination geometry in the active site of SorT.

https://doi.org/10.7554/eLife.09066.005
BondDistance (Å)
Mo-S1 (pterin)2.4
Mo-S2 (pterin)2.4
Mo-S (Cys 127)2.3
Mo=O1.7
Mo-OH/H2O1.9
Table 3

Spin Hamiltonian parameters for the Mo(V) center of SorT and various low and high pH forms of human, avian, plant and bacterial sulfite oxidases.

https://doi.org/10.7554/eLife.09066.007
SpeciesParameterXYZβoRef
SorTg1.949301.959971.98632-
A(95Mo)b20.536.053.926
A(1H)b,c3.54.04.80
SorAcg1.95411.96611.9914-dKlein, et al., 2013
Human SOg Low pH1.96461.97232.0023-Enemark, et al., 2010
A(1H)b11.477.107.71-Enemark, et al., 2010
Chicken SOg (Low pH)1.96581.97202.0037-Drew and Hanson, 2009
A(1H)b11.937.377.95-Drew and Hanson, 2009
g (High pH)1.95311.96411.9872Drew and Hanson, 2009
A. Thaliana SOg (Low pH)1.9631.9742.005-Enemark, et al., 2006
A(1H)b11.99.210.3--Enemark, et al., 2006
g (High pH)1.9561.9641.989-Enemark, et al., 2006
  1. aNon-coincident angle between g and A (rotation about x axis). bUnits 10-4 cm-1. c Two magnetically equivalent protons (I=1/2) were included in the computer simulated spectra. c95Mo hyperfine couplings were unresolved and the shoulders on gz were incorrectly attributed to 95Mo hyperfine resonances. dEuler angles were not determined.

Table 4

Data collection and processing parameters for analysis of the SorT/SorU complex in solution by Small Angle X-ray Scattering (SAXS).

https://doi.org/10.7554/eLife.09066.009
Data collection parameters
InstrumentSAXSess (Anton Paar)
Beam geometry10 mm slit
AH, LH (Å-1), GNOM beam geometry definition0.28, 0.12
q-range measured (Å-1)0.01-0.400
Exposure time (min)60 (4 x 15)
SorT2SorU2 concentration range (mg mL-1)2.75-5.5
Temperature (ºC)10
Structural parameters*
Rg (Å), I(0) (cm-1)
from Guinier (desmeared data) q*Rg < 1.3
30.8 ± 0.4, 0.223 ± 0.002
Rg (Å), I(0) (cm-1)
from P(r) (q-range 0.01 – 0.25 Å-1)
32.0 ± 0.3, 0.235 ± 0.002
dmax (Å) from P(r)110
Molecular mass determination*
Molecular mass Mrfrom Guinier I(0) (ratio with expected)108741 (0.984)
Molecular mass Mrfrom 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
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
SorT

31.3, 108
27.9, 99
Crystal structure compare to desmeared I(q) (χ-value)
SorT/SorU2/SorT (q-range 0.01 – 0.15 Å-1)
SorT (q-range 0.01 – 0.15 Å-1)

1.7
2.3
Results from 10 ab initio shape restorations. P1 symmetry:
Average molecular volume (Å3)
Normalised spatial distribution (NSD) and NSD variation
χ value for fit to desmeared data

140800
0.508 (0.008)
1.8
Software employed
Calculation of expected Mr, △ρ and υ valuesMULCh
Primary data reduction, I(q) vs q SAXSQuant 1D
DesmearingSAXSQuant
Guinier analysisPRIMUS
P(r) analysisGNOM
Model I(q) from crystal coordinatesCRYSOL
ab initio shape restorationsDAMMIN
3D graphics representationsPYMOL
  1. *Reported for 2.75 mg ml-1 measurement.

Table 5

Electron transfer parameters between SorT (Mo) and SorU (Fe) as calculated by PATHWAYS (Onuchic et al., 1992).

https://doi.org/10.7554/eLife.09066.011
Distance (Mo-Fe, Å)16.5 Å
Atomic packing density (ρ)0.97
Average decay exponential (β)0.97
Electronic coupling (HDA)3.4 x 10-4
Maximum ET rate (s-1)1.2 x 107
Table 6

Redox potential values for SorT and SorUa.

https://doi.org/10.7554/eLife.09066.014
ProteinCoupleEº (mV vs NHE)
SorTMoVI/V+110(±10)
MoV/IV-18(±10)
SorUFeIII/II+108 (±10)
SorU
(in the presence of SorT)
FeIII/II+111 (±10)
  1. aRedox potentials of SorT were determined by redox potentiometry, and SorU redox potentials by optical spectroelectrochemistry.

Table 7

Comparison of the protein-protein interfaces in the SorT/SorU and SorAB structures.

https://doi.org/10.7554/eLife.09066.015
ParameterSorT/SorUaSorABb
SorTSorUSorASorB
Average relative B factorc2)0.91.51.01.1
Buried surface area (Å2)d64469612541380
Interfacing residuesd31214633
Hydrogen-bonds630e
Salt-bridges12e
Shape complementarity statisticf0.630.77
  1. aThis work

  2. bPDB code 2BLF (Kappler and Bailey, 2005)

  3. cCalculated as the average for the protomer of interest divided by the average for the entire complex structure.

Data availability

The following data sets were generated
    1. McGrath AP
    2. Maher MJ
    (2015) Crystal structure of the sulfite dehydrogenase SorT from Sinorhizobium meliloti
    Publicly available at the Protien Data Bank (accession no. 4PW3).
    1. Laming EM
    2. McGrath AP
    3. Maher MJ
    (2015) Crystal structure of the c-type cytochrome SorU from Sinorhizobium meliloti
    Publicly available at the Protien Data Bank (accession no. 4PWA).

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