Characterization of Anammox Hydrazine Dehydrogenase, a Key N2-producing Enzyme in the Global Nitrogen Cycle.
Anaerobic ammonium-oxidizing (anammox) bacteria derive their energy for growth from the oxidation of ammonium with nitrite as the electron acceptor. N2, the end product of this metabolism, is produced from the oxidation of the intermediate, hydrazine (N2H4). Previously, we identified N2-producing hydrazine dehydrogenase (KsHDH) from the anammox organism Kuenenia stuttgartiensis as the gene product of kustc0694 and determined some of its catalytic properties. In the genome of K. stuttgartiensis, kustc0694 is one of 10 paralogs related to octaheme hydroxylamine (NH2OH) oxidoreductase (HAO). Here, we characterized KsHDH as a covalently cross-linked homotrimeric octaheme protein as found for HAO and HAO-related hydroxylamine-oxidizing enzyme kustc1061 from K. stuttgartiensis Interestingly, the HDH trimers formed octamers in solution, each octamer harboring an amazing 192 c-type heme moieties. Whereas HAO and kustc1061 are capable of hydrazine oxidation as well, KsHDH was highly specific for this activity. To understand this specificity, we performed detailed amino acid sequence analyses and investigated the catalytic and spectroscopic (electronic absorbance, EPR) properties of KsHDH in comparison with the well defined HAO and kustc1061. We conclude that HDH specificity is most likely derived from structural changes around the catalytic heme 4 (P460) and of the electron-wiring circuit comprising seven His/His-ligated c-type hemes in each subunit. These nuances make HDH a globally prominent N2-producing enzyme, next to nitrous oxide (N2O) reductase from denitrifying microorganisms.
From the Department of Microbiology, Institute for Water and Wetland Research, Radboud University Nijmegen, 6525 AJ Nijmegen, The Netherlands.
Wouter J
WJ
Maalcke
From the Department of Microbiology, Institute for Water and Wetland Research, Radboud University Nijmegen, 6525 AJ Nijmegen, The Netherlands.
Joachim
J
Reimann
the Department of Biotechnology, Delft University of Technology, 2628 BC Delft, The Netherlands.
Simon
S
de Vries
the Centre for Molecular and Structural Biochemistry, School of Chemistry and School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom, and.
Julea N
JN
Butt
the Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, 69120 Heidelberg, Germany.
Andreas
A
Dietl
From the Department of Microbiology, Institute for Water and Wetland Research, Radboud University Nijmegen, 6525 AJ Nijmegen, The Netherlands.
Nardy
N
Kip
the Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, 69120 Heidelberg, Germany.
Ulrike
U
Mersdorf
the Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, 69120 Heidelberg, Germany.
Thomas R M
TR
Barends
From the Department of Microbiology, Institute for Water and Wetland Research, Radboud University Nijmegen, 6525 AJ Nijmegen, The Netherlands, the Department of Biotechnology, Delft University of Technology, 2628 BC Delft, The Netherlands.
Mike S M
MS
Jetten
From the Department of Microbiology, Institute for Water and Wetland Research, Radboud University Nijmegen, 6525 AJ Nijmegen, The Netherlands.
Jan T
JT
Keltjens
From the Department of Microbiology, Institute for Water and Wetland Research, Radboud University Nijmegen, 6525 AJ Nijmegen, The Netherlands, bkartal@mpi-bremen.de.
Boran
B
Kartal
D064751:Ammonium Compounds / Q000737:chemistry* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D006834:Hydrazines / Q000737:chemistry* / Q000378:metabolism; D009584:Nitrogen / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D061328:Planctomycetales / Q000201:enzymology* / Q000235:genetics
D064751:Ammonium Compounds; D001426:Bacterial Proteins; D006834:Hydrazines; C029424:hydrazine; D010088:Oxidoreductases; D009584:Nitrogen
anaerobic ammonium oxidation; electron paramagnetic resonance (EPR); enzyme kinetics; enzyme purification; hydrazine; nitrogen metabolism; octaheme proteins; reactive nitrogen species (RNS)