A 192-heme electron transfer network in the hydrazine dehydrogenase complex

Abstract

Anaerobic ammonium oxidation (anammox) is a major process in the biogeochemical nitrogen cycle in which nitrite and ammonium are converted to dinitrogen gas and water through the highly reactive intermediate hydrazine. So far, it is unknown how anammox organisms convert the toxic hydrazine into nitrogen and harvest the extremely low potential electrons (-750 mV) released in this process. We report the crystal structure and cryo electron microscopy structures of the responsible enzyme, hydrazine dehydrogenase, which is a 1.7 MDa multiprotein complex containing an extended electron transfer network of 192 heme groups spanning the entire complex. This unique molecular arrangement suggests a way in which the protein stores and releases the electrons obtained from hydrazine conversion, the final step in the globally important anammox process.

Fig. 1. HDH complex.

(A and B) Negative-stain electron micrographs of K. stuttgartiensis HDH alone in the absence and presence (300 mM) of KCl. (C and D) B. fulgida HDH alone, without salt, and with 300 mM KCl. (E) Crystal structure of the K. stuttgartiensis HDH/Kustc1130 assembly. HDH trimers are shown in green, with the monomers of one trimer shown in different shades of green. Assembly factor molecules are shown in beige. (F and G) K. stuttgartiensis HDH supplemented with the assembly factor Kustc1130 without salt and with 300 mM KCl. (H and I) B. fulgida HDH supplemented with the assembly factor Broful2728 without salt and with 300 mM KCl.

Fig. 2. Electron transfer network in HDH.

(A) Entire HDH assembly, with one trimer (front, red circle) shown in different shades of green. (B) Heme network in a trimer of HDH. The eight heme groups of each monomer form a ring-like relay system for electrons, connecting the active site heme 4 moieties to the exit sites for electrons at heme 1 as in a typical HAO-like enzyme. (C) Heme networks of two individual trimers in the HDH complex. Heme 1 of the one trimer is in close proximity to a heme 1 of the other trimer, likely allowing efficient electron transfer. Their edge-to-edge distance is indicated. (D) Proposed network of heme groups in the HDH complex. Each heme group is represented by its iron atom, shown as a red sphere or a blue sphere in case of an active site heme 4 iron. The surface of the HDH complex is shown as a black outline. The heme network approximates a truncated cube. (E). Schematic of part of the heme network in the HDH complex. Active site hemes (labeled “4”) are shown in solid blue, and other heme groups are shown as open black symbols. A possible path for electrons from one active site to a distant trimer in the complex is indicated by the red line.

Fig. 3. Details of the HDH complex structure.

(A) Close-up of the active site. Three monomers, shown in different colors, contribute side chains to the active-site cavity, such as the conserved Asp²⁵⁵/His²⁵⁶ pair from one monomer and the N-terminal Val^33′ from another. The active-site heme 4 is bound covalently to Tyr^462″ from a third monomer, as well as to the conserved Cys²⁰² in addition to the two cysteines of the heme-binding motif (in the background). (B) Sliced view of the HDH complex. Heme groups 3 of each monomer are solvent-exposed just inside the holes, leading to the central cavity in the complex.