Rieske Iron-Sulfur Cluster Proteins from an Anaerobic Ammonium Oxidizer Suggest Unusual Energetics in their Parent Rieske/cytochrome b complexes

Anaerobic ammonium-oxidizing (anammox) bacteria employ a unique, hydrazine-based pathway to obtain energy from nitrite and ammonium. These organisms express distinct Rieske/ cytochrome b complexes of which the exact function in anammox metabolism is unclear, but which has been proposed to include the generation of NAD(P)H. This would require energetics and structural features unusual for such complexes. Here we present crystal structures and electrochemical investigations of the Rieske subunits of two of these complexes from the anammox organism Kuenenia stuttgartiensis, Kuste4569 and Kustd1480. Both proteins display high redox potentials (>+300 mV), which can be in part explained by their crystal structures and which fit perfectly in the energetic scheme of the proposed NAD(P)H generation mechanism. Moreover, AlphaFold3 models of the parent complexes trace out a path for the electrons required for NAD(P) production, which includes a proposed, novel b-type heme in the membrane-bound part of the complex.