Figures and data in Structural basis of interprotein electron transfer in bacterial sulfite oxidation

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Tables

9 figures and 7 tables

Figures

Figure 1

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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

Figure 2

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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

Figure 3

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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

Figure 4

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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

Figure 5

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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

Figure 6

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Redox analyses of the SorT protein and the SorT/SorU complex.

(A) Plot of EPR intensity (I_p) at 343 mT (from Mo^V form of SorT) as a function of solution redox potential (E mV vs NHE). The solid line is a fit to the equation $I (E) = \frac{I_{p}}{1 = 10^{(E - E_{1}) / 59} + 10^{(E_{2} - E) / 59}}$ using the potentials E₁ = Mo^VI/V = +110(±10) mV and E₂ Mo^V/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 $A b s = \frac{(ε_{o x} 10^{(E - E') / 59}) + ε_{r e d})}{1 + 10^{(E - E') / 59}} C_{t o t}$ where the extinction coefficients refer to the oxidized and reduced forms of the protein and *Abs* is the absorbance at this same wavelength. *c_tot* 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

Figure 7

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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

Figure 8

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Redox mediators employed in optical spectroelectrochemistry experiments.
https://doi.org/10.7554/eLife.09066.017

Appendix 2-figure 1

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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), *d_max* 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

	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)
<I/σ(I)>	6.7 (2.1)	8.9 (1.6)	8.8 (1.7)
R_merge (%)^b	15.9 (66.7)	13.3 (76.6)	13.9 (76.6)
Refinement
Reflections in working set	74024	22113	17781
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
Metal atoms	4	4	2
Water atoms	376	227	63
Other atoms	108	168	67
R_work (%)^c	20.8 (31.7)	19.2 (30.8)	21.1 (30.2)
R_free (%)^d	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
<B> (Å^2)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 R_merge = ∑_hkl ∑_i| I_i (hkl) - <I(hkl)> |/∑_hkl ∑_i I_i (hkl)
^c R_work = ∑_h | F_obs – F_calc |/∑_hF_obs
^d Calculated as for R_work using 10% of the diffraction data that had been excluded from the refinement
^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

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

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

Species	Parameter	X	Y	Z	β^o	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
SorA^c	g	1.9541	1.9661	1.9914	-^d	Klein, et al., 2013
Human SO	g Low pH	1.9646	1.9723	2.0023	-	Enemark, et al., 2010
	A(¹H)^b	11.47	7.10	7.71	-	Enemark, et al., 2010
Chicken SO	g (Low pH)	1.9658	1.9720	2.0037	-	Drew and Hanson, 2009
	A(¹H)^b	11.93	7.37	7.95	-	Drew and Hanson, 2009
	g (High pH)	1.9531	1.9641	1.9872		Drew and Hanson, 2009
A. Thaliana SO	g (Low pH)	1.963	1.974	2.005	-	Enemark, et al., 2006
	A(¹H)^b	11.9	9.2	10.3	--	Enemark, et al., 2006
	g (High pH)	1.956	1.964	1.989	-	Enemark, et al., 2006

^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 g_z 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
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)
SorT₂SorU₂ concentration range (mg mL^-1)	2.75-5.5
Temperature (ºC)	10
Structural parameters^*
R_g (Å), I(0) (cm^-1) from Guinier (desmeared data) qR_g* < 1.3	30.8 ± 0.4, 0.223 ± 0.002
R_g (Å), I(0) (cm^-1) from P(r) (q-range 0.01 – 0.25 Å^-1)	32.0 ± 0.3, 0.235 ± 0.002
d_max (Å) from P(r)	110
**Molecular mass determination^***
Molecular mass M_rfrom Guinier I(0) (ratio with expected)	108741 (0.984)
Molecular mass M_rfrom P(r) I(0) (ratio with expected)	114593 (1.037)
SorT₂SorU₂ parameters calculated from sequence and chemical composition
Molecular volume (Å³)	134385
Molecular weight M_r (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 R_g, d_max (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 M_r, △ρ 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
3D graphics representations	PYMOL

^*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 (H_DA)	3.4 x 10^-4
Maximum ET rate (s^-1)	1.2 x 10⁷

Table 6

Redox potential values for SorT and SorU^a.

https://doi.org/10.7554/eLife.09066.014

Protein	Couple	Eº (mV vs NHE)
SorT	Mo^VI/V	+110(±10)
SorT	Mo^V/IV	-18(±10)
SorU	Fe^III/II	+108 (±10)
SorU (in the presence of SorT)	Fe^III/II	+111 (±10)

^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

Parameter	SorT/SorU^a		SorAB^b
	SorT	SorU	SorA	SorB
Average relative B factor^c (Å²)	0.9	1.5	1.0	1.1
Buried surface area (Å^2)d	644	696	1254	1380
Interfacing residues^d	31	21	46	33
Hydrogen-bonds	6		30^e
Salt-bridges	1		2^e
Shape complementarity statistic^f	0.63		0.77

^aThis work
^bPDB code 2BLF (Kappler and Bailey, 2005)
^cCalculated as the average for the protomer of interest divided by the average for the entire complex structure.
^d(Krissinel and Henrick, 2007)
^eTaken from(Kappler and Bailey, 2005)
^f(Lawrence and Colman, 1993)