# Rieske Iron–Sulfur Cluster Proteins From an Anaerobic Ammonium Oxidizer Suggest Unusual Energetics in Their Parent Rieske/Cytochrome b Complexes

## TL;DR

This study explores unique Rieske proteins in anammox bacteria, revealing their role in energy generation through unusual redox properties and structural features.

## Contribution

The paper provides crystal structures and electrochemical data of Rieske subunits from anammox bacteria, supporting a novel NAD(P)H generation mechanism.

## Key findings

- Rieske subunits from Kuenenia stuttgartiensis show high redox potentials (> + 300 mV).
- Structural and electrochemical data align with the proposed NAD(P)H generation mechanism.
- AlphaFold3 models suggest a novel b-type heme in the electron transfer pathway.

## Abstract

Anaerobic ammonium‐oxidizing (anammox) bacteria employ a unique, hydrazine‐based pathway to obtain energy from nitrite and ammonium. These organisms possess distinct Rieske/cytochrome b complexes whose precise role in anammox metabolism remains unclear, but which have 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 into 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)H production, which includes a proposed, novel b‐type heme in the membrane‐bound part of the complex.