Photon Factory Activity Report 2014 #32 (2015) B

BL-5A, BL-17A/2013G122

High-resolution crystal structures of the solubilized domain of porcine cytochrome b5
Yu Hirano and Taro Tamada*
QuBS, JAEA, Tokai 319-1195, Japan
1 Introduction
Mammalian cytochrome b5 has multiple electrontransfer partners that function in various electron-transferreactions. Extensive mutational studies have beenreported based on the crystal structure of bovine
cytochrome b5. However, high-resolution structures havenot been reported for both the oxidized and reduced statesof cytochrome b5. We have determined four crystal
structures at sub-angstrom resolutions in two crystal
forms for both the oxidized and reduced states [1].
2 Experiment
Two crystal forms (form 1 and 2) were obtained under
the conditions containing polyethylene glycol as aprecipitant. Crystals of the oxidized form were obtained
under an aerobic condition and crystals of the reduced
form were obtained under an anaerobic condition. The
concentrations of the polyethylene glycol were increased
to collect diffraction data at 100 K. The crystals of theoxidized form were flash-cooled in a nitrogen-gas stream.
The crystals of the reduced form were cooled in liquid
nitrogen under an anaerobic condition.
Diffraction data sets were collected on the BL-5A and
BL-17A beamlines. Two data sets for high-resolution andlow-resolution were separately collected from a single
crystal at different positions. For the high-resolution datasets, the positions at which the crystals were exposed toX-rays were changed in order to restrict the maximum
dose for each exposure position. For the low-resolution
data sets, the crystals were exposed to attenuated X-rays
to avoid overloading the CCD detectors.
3 Results and Discussion
The crystal structures were determined at resolutions of0.83 Å (oxi, form1), 0.93 Å (oxi, form 2), 0.76 Å (red,form1) and 0.95 Å (red, form 2) (Table 1). The final
models contained 20-28 multiple conformations ofamino-acid residues. 17-48% of the hydrogen atoms in
the amino-acid residues could be included in the models(Fig. 1a).
Unrestrained refinement for bond angles and lengths
indicated that the 7-propionate group of the heme waspossibly protonated in the reduced form 2 structure.
Form 2 crystals were obtained at pH 5.5, which is lower
than the experimentally measured pKa value of 5.9. On
the other hand, the 7-propionate group was in a charged
state in the oxidized form 2 structure. The protonation
states in the form 2 structures are consistent withelectrostatics calculations, in which heme oxidation has
been reported to be coupled to some proton loss.
Structural changes depending on the redox states wereobserved in the hydrogen bond distances from the heme
axial ligand His68 to Phe63 between the form 1 structures(Fig. 1b). In addition, water molecules form hydrogenbonds to both Phe63 and His68. The hydrogen-bonddistances of the water molecules are different between theoxidized and reduced forms of the form 1 structures.
These results indicate that the hydrogen-bond network
around His68 might be involved in regulating the redox
state of the heme.

Fig. 1: (a) Electron densities (hydrogen omit maps, 1.5 σ)and (b) hydrogen bonds around the heme axial ligand His
68.
Table 1: Diffraction data statistics
Oxi-1
Oxi-2
Red-1
Red-2
Beamline
BL-17A BL-17A BL-5A
BL-5A
Wavelength
0.91
0.91
0.800.90(Å)
50-0.83 50-0.93 50-0.76 50-0.95
Resolution(0.84(0.95(0.77(0.97(Å)
0.83)
0.93)
0.76)
0.95)
Unique72,78963,82499,19658,020reflections
Completeness
98.2
98.8
99.8
95.8
(%)
(82.3)(96.7)(97.3)(87.0)
8.9
6.8
7.1
8.4
Rr.i.m. (%)(37.4)(43.5)(38.6)(43.9)
59.8
50.9
67.4
54.8
<I>/<(I)>
(7.6)
(3.5)
(4.1)
(2.3)
Values in parentheses represent the highest resolution
shells.
References[1] Y. Hirano, S. Kimura and T. Tamada, Acta Cryst. D71,
1572 (2015).
* tamada.taro@jaea.go.jp