Structure of hydrazine synthase, an electron transfer complex

Daisuke HIRA

Department of Applied Life Science, Faculty of Biotechnology
and Life Science, Sojo University

Research aims
Wastewater treatment using anaerobic ammonium oxidation (anammox) bacteria is
an innovative nitrogen treatment technology, and its application to actual wastewater
has begun. In the anammox reaction, nitrite is reduced by ammonia as the electrondonor, after passing through hydrazine (N2H4) as a reaction intermediate, and nitrogenmolecules are produced (NH4+ + NO2- → N2 + 2H2O). However, the reactionmechanism is not fully understood. In particular, the N2H4 synthesis reaction is foundonly in anammox bacteria, and its details were completely unknown until relativelyrecently. In 2011, there was a report on hydrazine synthase (HZS, a heterotrimeric heme
protein) as an enzyme for the synthesis of N2H4 from NH3 and nitrogen monoxide(NO)1). In the present study, the reaction product N2H4 was not detected directly, andthe reaction rate was very low. However, we purified another heme protein, NaxLS (aheterodimer consisting of NaxL and NaxS subunits)2), and determined its

three-dimensional structure. The structure revealed that the heme iron of NaxLS iscoordinated by His/Cys, and indicated that NaxLS works as a specific electron transfer
protein within the anammox reaction. We attempted a hydrazine synthesis reaction
using HZS and NaxLS, and detected the production of N2H4 from NH3 and NO assubstrates. It was proposed that the electron transfer reaction from NaxLS to HZS is anindispensable step in hydrazine synthesis. From these results, NaxLS was suspected tofunction as a physiological electron transfer protein for HZS. In the present study, weattempted to reveal the structure of the HZS-NaxLS electron transfer complex, using
X-ray crystal structure analysis.

Methods
HZS and NaxLS were purified from an enrichment culture of the anammox
bacterium strain KSU-1, via column chromatography. Using a mixture of both proteins,
crystallization conditions were determined via the vapor diffusion method.
Approximately 200 conditions were investigated, using a commercial screening kit. The
obtained crystals were analyzed using SDS-PAGE, and complex crystals were frozen
and stored in liquid nitrogen. Diffraction data were collected at the SPring-8 BL44XU
and BL41XU, and were integrated and scaled using HKL2000 and XDS. The structureof the HZS-NaxLS protein complex was solved using a combination of Fe-SAD
phasing and the molecular replacement method.
kDaResults
After performing

MWmarker

97.4
66
43

crystallization procedures

HZS

31

with a mixture of the HZSand NaxLS proteins, single

21.5

crystals of the complex of14.5

both proteins were obtained,
using pentaerythritol
ethoxylate andpolyvinylpyrrolidone as theprecipitants (Figure 1). X-ray

NaxLS0.3 mm

Figure 1. Crystals of the HZS-NaxLS protein
complex (left) and SDS-PAGE results of the crystals(right).

diffraction data for thesecrystals were then obtained ata resolution of 2.8 Å. Using
the molecular replacement
method and the crystal
structure of HZS3), we wereable to determine the crystal
structure of the HZS-NaxLS
protein complex (Figure 2).
HZS formed a dimer of theheterotrimer αβγ, and one

Figure 2. Crystal structure of the HZS-NaxLS protein

NaxLS molecule was boundcomplex. The red dashed line indicates the NaxLSmolecule.

to the heterotrimer (αβγ) of HZS. In addition, we determined that a heme coordinated bythe His/Cys of the NaxL subunit of NaxLS, and a 6 coordinated heme of the γ subunit ofHZS, were located at a distance of 12 Å. We determined that an intermolecular electrontransfer reaction is possible through these hemes.
Conclusion
N2H4 synthesis from NH3 and NO requires the transfer of 3 electrons. From therevealed structure of the hydrazine synthase complex consisting of HZS and NaxLS, andfrom biochemical experimental results, we hypothesized that a two-electron-transfer
reaction from NaxLS to HZS is an essential step in the hydrazine synthesis reaction. Our
next goal will be to elucidate the effects of formation of this protein complex on themechanisms of hydrazine synthesis reaction, based on the known structure of thiscomplex.

References
1) Kartal B et al., (2011) Molecular mechanism of anaerobic ammonium oxidation. Nature. 479:
127–130.

2) Ukita S et al., (2010) A heterodimeric cytochrome c complex with a very low redox potentialfrom an anaerobic ammonium-oxidizing enrichment culture. FEMS Microbiol Lett. 313:61–67.
3) Dietl A, et al., (2015) The inner workings of the hydrazine synthase multiprotein complex.
Nature. 527: 394–397.