Crystallization and preliminary crystallographic studies of FoxE from Rhodobacter ferrooxidans SW2, an FeII oxidoreductase involved in photoferrotrophy

crystallization communications

1106

doi:10.1107/S174430911203271X

ActaCryst.

(2012). F

, 1106–1108

Acta Crystallographica Section F

Structural Biology

and Crystallization

Communications

ISSN 1744-3091

Crystallization and preliminary crystallographic

studies of FoxE from

Rhodobacter ferrooxidans

SW2, an Fe

oxidoreductase involved in

photoferrotrophy

L. Pereira,

I. H. Saraiva,

R. Coelho,

D. K. Newman,

R. O. Louro

and C. Fraza

Instituto de Tecnologia Quı

mica e Biolo

gica,

Universidade Nova de Lisboa, Apartado 127,

2781-901 Oeiras, Portugal, and

California

Institute of Technology/Howard Hughes Medical

Institute, 1200 East California Boulevard, Mail

Code 147-75, Pasadena, CA 91125, USA

Correspondence e-mail: frazao@itqb.unl.pt

Received 22 June 2012

Accepted 18 July 2012

FoxE is a protein encoded by the

foxEYZ

operon of

Rhodobacter ferrooxidans

SW2 that is involved in Fe

-based anoxygenic photosynthesis (‘photoferro-

trophy’). It is thought to reside in the periplasm, where it stimulates light-

dependent Fe

oxidation. It contains 259 residues, including two haem

-binding

motifs. As no three-dimensional model is available and there is no structure with

a similar sequence, crystals of FoxE were produced. They diffracted to 2.44 A

resolution using synchrotron radiation at the Fe edge. The phase problem was

solved by SAD using

SHELXC

and the experimental maps confirmed the

presence of two haems per molecule.

1. Introduction

FoxE is a di-haem

-type cytochrome encoded by the

foxEYZ

operon

from the bacterium

Rhodobacter ferrooxidans

SW2, a bacterium that

is capable of photoferrotrophy. This is a metabolic strategy that is

characterized by the utilization of ferrous iron (Fe

) oxidation as the

sole source of electrons for photosynthesis (Widdel

et al.

, 1993). The

ferric iron (Fe

III

) resulting from this metabolism precipitates as ferric

(hydr)oxides at the neutral pH at which this organism grows. This

ancient form of photosynthesis (Xiong

et al.

, 2000) is hypothesized to

have catalyzed the deposition of ancient sedimentary deposits known

as banded iron formations in early phases of the history of the Earth

(Kappler

et al.

, 2005).

Heterologous expression of the

fox

operon in the bacterium

R. capsulatus

SB1003 resulted in enhanced photosynthetic Fe

oxidation activity, which suggested that the products of this operon

are required for photoferrotrophy (Croal

et al.

, 2007). That the

expression of

foxE

alone is enough to enhance photosynthetic Fe

oxidation indicates that this cytochrome is responsible for the

-oxidation step in this metabolism. Previous functional char-

acterization of FoxE (Saraiva

et al.

, 2012) demonstrated that it is

thermodynamically and kinetically capable of oxidizing Fe

in vitro

In-depth characterization of the proteins involved in photoferro-

trophy is required in order to understand this ancient form of

photosynthesis which is likely to have had a profound impact on the

geochemical evolution of the Earth in the past.

2. Materials and methods

2.1. Protein expression and purification

FoxE was cloned, expressed and purified as described by Saraiva

et al.

(2012). The protein purity was checked by SDS–PAGE and the

pure protein was concentrated using Vivaspin concentrators with a

30 kDa cutoff membrane, leading to a final protein solution consisting

of 15–18 mg ml

FoxE in 5 m

potassium phosphate buffer pH 7.0.

2.2. Protein crystallization

Initial FoxE crystallization screens were performed at room

temperature (293 K) by the vapour-diffusion method with commer-

cially available solution kits in a Mini-Bee MicroSys 4000XL

Cartesian Dispensing Systems robot (Genomic Solutions, USA).

Drops were produced by dispensing 100 nl protein solution plus

100 nl precipitant solution and were equilibrated against 100

m

#

2012 International Union of Crystallography

precipitant solution; they were examined under a magnifying glass to

detect possible crystallization hints. These were then further opti-

mized in microlitre-scale drops by variation of the initial conditions,

by the use of crystallization additives and by attempting seeding

experiments.

Two crystallization screens, Structure Screen 1 and Structure

Screen 2 from Molecular Dimensions Ltd (Suffolk, England), were

initially tried. No crystallization hits were detected among those 2

48 trials, but precipitation was observed in

50% of the drops with

solutions containing PEG and in

60% of the drops with pH below

6.5. MacroSol, JCSG-plus and Stura Footprints screens from Mole-

cular Dimensions were then tried. Three distinct crystal shapes were

obtained from these 192 conditions: needle-like crystals were

obtained using solution 2.16 of MacroSol [0.1

HEPES pH 7.5,

2.0

ammonium sulfate, 5%(

) PEG 400], polyhedral crystals with

similar shapes were obtained using four solutions from JCSG-plus

that contained PEG, namely solution 1.2 [0.1

sodium citrate pH

5.5, 20%(

) PEG 3K], solution 1.45 [0.17

ammonium sulfate,

25.5%(

) PEG 4K, 15% glycerol], solution 1.21 [0.1

citrate pH 5,

20%(

) PEG 6K] and solution 2.45 [0.2

lithium sulfate, 0.1

bis-Tris pH 5.5, 25%(

) PEG 3350], and agglomerated polycrystals

were obtained using Stura Footprints solution B6 (1.32

sodium/

potassium phosphate pH 7). The conditions that delivered the best-

looking crystals were optimized on the microlitre scale, aiming to

control nucleation and to increase the crystal size. However, the use

of JCSG-plus solution 1.2 could not be reproduced and solution 1.45

only produced crystals at 303 K. The use of PEGs of sizes 2–6K at

5–20%, the addition of 5 or 15% glycerol, the removal of sodium

citrate, the use of drops with different volumes of protein and

precipitant (2:1.5, 2:1, 2.2:0.8 and 2.3:0.7), the addition of silicone,

paraffin or PEG 400 to control vapour diffusion and the use of macro-

seeding were attempted. These attempts revealed that the crystals

were often not reproducible and showed a gel-like consistency within

4–7 d, therefore not being amenable to X-ray diffraction.

We proceeded with the non-PEG lead, Stura Footprints solution

B6 based on phosphate precipitant. Although its microlitre scale-up

was reproducible at all temperatures tested (277, 293 and 303 K),

diffraction experiments showed that the crystals were disordered.

Crystallization conditions at 293 K were then varied using PEGs of

sizes 2–6K, 10–36%(

) PEG concentration, 0.1–1.4

phosphate

concentration, pH values in the range 5–7.2 and different (1:2, 1:1 and

2:1) dispensing ratios. Better crystals grew in 0.2

sodium/potassium

phosphate pH 5, 20%(

) PEG 5K, but degraded over time and did

not produce usable diffraction. In order to sample wider crystal-

lization conditions using phosphate precipitant, the Additive Screen

from Hampton Research (Aliso Viejo, USA) was also used, restricted

to its 80 nonvolatile conditions. The best crystals were obtained at

293 K in sitting drops consisting of 1

m

l15mgml

protein solution

plus 1

m

l precipitant solution (1.2

sodium/potassium phosphate

pH 7, 50 m

copper chloride) equilibrated against 500

m

l precipitant

solution in the well. Red polyhedral crystals reached dimensions of

up to approximately 0.1

0.1

0.02 mm (Fig. 1). Crystal cryo-

protection was accomplished by soaking the crystals in mother liquor

complemented with 25%(

) glycerol.

2.3. X-ray diffraction data collection and crystal phase-problem

solution

The diffraction of a cryostabilized crystal was measured near the

iron absorption edge using an ADSC Q315R detector at station

ID23-1 of the European Synchrotron Radiation Facility (ESRF),

Grenoble, France. Diffraction images from two 360

crystal rotations,

interleaved by a crystal translation in order to start each diffraction

run from an unexposed crystal region, were integrated and scaled

with

XDS

(Kabsch, 2010) and the resulting two sets of intensities

were merged together with

XPREP

(Bruker).

The phase problem was solved by single-wavelength anomalous

diffraction (SAD) using the

HKL

MAP

GUI (Pape & Schneider,

2004; Sheldrick, 2008), which (i) relies on

SHELXC

to analyse the

data, estimate anomalous difference factors and produce initial phase

shifts for reflections corresponding to the largest normalized anom-

alous differences, (ii) uses

SHELXD

to determined the anomalous

heavy-atom substructure and (iii) uses

SHELXE

to produce iterative

phase improvement by density modification, including automated

poly-Ala chain tracing (Sheldrick, 2010).

3. Results and discussion

Despite several attempts to optimize the initial FoxE crystallization

conditions from PEG- or phosphate-containing solutions, the crystals

were not always reproducible; their diffraction indicated disordered

crystal packing and showed severe intensity reduction within a few

days, when the crystals became gel-like red polyhedra. The addition

of copper chloride as an additive to the mother liquor, however, led

to increased crystal stability and to increased diffraction capability.

crystallization communications

ActaCryst.

(2012). F

, 1106–1108

Pereira

etal.

FoxE

1107

Figure 1

Crystallization drop of FoxE showing red well shaped polyhedral crystals with

approximate dimensions of 0.1

0.1

0.02 mm.

Table 1

Crystallographic and diffraction data statistics.

Values in parentheses are for the highest resolution shell.

ESRF beamline

ID23-1

Wavelength (A

)

1.73925

Resolution (A

)

57.83–2.44 (2.54–2.44)

Space group

Unit-cell parameters (A

)

= 112.77,

= 143.54

No. of measured reflections

1293342

No. of unique reflections

38526 (3591)

Multiplicity

32.2 (6.3)

Mean

(

)

28.3 (1.9)

p.i.m.

† (%)

2.2 (25.0)

merge

‡ (%)

13.6 (70.7)

Completeness (%)

96.1 (77.1)

Solvent content (%)

52.3

Wilson

factor (A

)

49.1

†

p.i.m.

P

hkl

P

hkl

Þh

hkl

Þij

P

hkl

P

hkl

, where

is the

data multiplicity,

(

hkl

) is the observed intensity and

(

hkl

)

is the average intensity of

multiple observations from symmetry-related reflections. It is an indicator of the

precision of the final merged and averaged data set (Weiss, 2001).

‡

merge

P

hkl

P

hkl

Þh

hkl

Þij

P

hkl

P

hkl

, where

(

hkl

) is the observed intensity and

(

hkl

)

is the average intensity of multiple observations from symmetry-related

reflections.

FoxE crystallized in space group

21 or

21, with unit-cell

parameters

= 112.77,

= 143.54 A

, and the crystals diffracted to

2.44 A

resolution (see Table 1 for further crystallographic details and

diffraction data statistics).

The diffraction data measured using X-rays at the Fe absorption

edge contained significant anomalous signal up to 3.7 A

resolution,

where the self-anomalous correlation coefficient remained above

30%. The probability distribution of the Matthews coefficient for

2.5 A

resolution indicated a 74% probability of four FoxE molecules

in the asymmetric unit, in contrast to a 17% probability of an

asymmetric unit containing only three molecules (Kantardjieff &

Rupp, 2003). As the FoxE sequence contains two haem

cytochrome

CH motifs (Croal

et al.

, 2007),

SHELXD

was set to search for

eight Fe atoms. However, only six anomalous atoms were found with

occupancies in the range 0.8–1.0; further hypothetical sites refined to

occupancies below 0.2 and were discarded. Accordingly, the FoxE

crystal contained three molecules in the asymmetric unit, corre-

sponding to a calculated crystal solvent content of 52% (Matthews,

1968).

SHELXE

was run twice. The first run, using phase information

from the six-Fe-atom substructure, solved the space-group hand

ambiguity, produced initial experimental maps, traced a 641 poly-Ala

residue model in 35 chains and listed a set of further anomalous sites.

These were examined with

Coot

(Emsley

et al.

, 2010) to determine the

12 thioether sites that attach the six haems to the three FoxE mole-

cules. A second

SHELXE

run was thus performed including the

phases from a further 12 S atoms and produced the final experimental

maps (Fig. 2), in which 678 poly-Ala residues of the expected 777

were traced in a model with 18 chains and a correlation coefficient of

35%.

Three-dimensional structure determination and refinement of

FoxE is in progress in order to unravel the molecular arrangement of

this

-type cytochrome with unprecedented primary structure that is

capable of oxidizing iron

in vitro

in a pH-dependent manner that

appears to be designed to prevent incrustation of the cells by ferric

iron.

The authors gratefully acknowledge the ESRF, Grenoble, France

for provision of synchrotron radiation and thank Susana Gonc

̧ alves,

ITQB–UNL, Portugal for diffraction data collection. G. M. Sheldrick

is thanked for valuable discussions and for providing the

SHELXE

program. This work was funded by projects MIT-Pt/BS-BB/1014/2008

from the MIT-Portugal Program and PTDC/EBB-BIO/098352/2008

from FCT, Portugal. IHS is the recipient of a PhD grant from FCT

(SFRH/BD/36582/2007). DKN is an HHMI Investigator.

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crystallization communications

1108

Pereira

etal.

FoxE

ActaCryst.

(2012). F

, 1106–1108

Figure 2

Experimental SAD electron density (blue mesh, 0.7

) and anomalous density (red

mesh, 5

) of a representative region of FoxE showing a haem and its ligands. (

)

and (

) show views with the haem plane perpendicular and parallel to the paper,

respectively. The Fe-atom localization is recognisable in the anomalous map at the

haem centre and the thioether S atoms are recognisable at the haem periphery.

The maps were produced with

SHELXE

(Sheldrick, 2010) and the figures were

produced with

PyMOL

(DeLano, 2002).