A di-iron protein recruited as an Fe[II] and oxygen sensor for bacterial chemotaxis functions by stabilizing an iron-peroxy species

Abstract

Many bacteria contain cytoplasmic chemoreceptors that lack sensor domains. Here, we demonstrate that such cytoplasmic receptors found in 8 different bacterial and archaeal phyla genetically couple to metalloproteins related to β-lactamases and nitric oxide reductases. We show that this oxygen-binding di-iron protein (ODP) acts as a sensor for chemotactic responses to both iron and oxygen in the human pathogen Treponema denticola (Td). The ODP di-iron site binds oxygen at high affinity to reversibly form an unusually stable μ-peroxo adduct. Crystal structures of ODP from Td and the thermophile Thermotoga maritima (Tm) in the Fe[III]2-O22−, Zn[II], and apo states display differences in subunit association, conformation, and metal coordination that indicate potential mechanisms for sensing. In reconstituted systems, iron-peroxo ODP destabilizes the phosphorylated form of the receptor-coupled histidine kinase CheA, thereby providing a biochemical link between oxygen sensing and chemotaxis in diverse prokaryotes, including anaerobes of ancient origin.

Atypical chemoreceptor arrays accommodate high membrane curvature

“…Previous experiments demonstrate that a protein called o xygen-binding d iiron p rotein (ODP) functions as an oxygen sensor for chemotaxis in Td , however the study did not determine if ODP is an integral component of the array 11 . This protein is genetically coupled to a soluble receptor, TDE2496, with unclear signaling capabilities.…”

“…To determine if the presence of ODP (TDE2498) and its cognate receptor (TDE2496) impacts array architecture or integrity, we conducted cryo-ET with two Td gene knock-out strains, Δ2498 and Δ2498Δ2496 11 . Importantly, reverse-transcription PCR confirms that deletion of ODP does not impact transcription of TDE2496 11 . The sub-volume averages of these strains reveal distinct differences in array densities compared to the wild-type (WT) strain (Fig.…”

“…To reduce redundancy in sequence datasets we used CD-HIT v4.6 8 with unaligned sequences and Jalview 9 with aligned sequences. RAxML v8.2.10 10 was used to perform phylogenetic reconstructions, and low support branches in the phylogenetic trees were collapsed with TreeCollapseCL4 11 . Sequence logos were built using Weblogo 3.7 12 .…”

“…We demonstrate that a CheR-like fusion domain in a variant CheW is key for maintaining the structural integrity of the arrays. Furthermore, cryo-ET analysis of Td cells lacking the previously characterized oxygen sensor ODP demonstrates significant changes in CheA mobility 11 .…”

Visualizing the superfamily of metallo-β-lactamases through sequence similarity network neighborhood connectivity analysis

“…A typical His-to-Glu mutation appears at motif 2 of FDPs, located at the interface between the isoalloxazine and di-iron moieties, which likely contributes to hold the more acidic Fe(III) species. An unusual metal coordination set is found in Thermotoga maritima diiron oxygen sensor ODP [31], where the third histidine of motif 2 is replaced by a glutamine at motif 5. Finally, the divergent class-C type-2 FDPs from Synechocystis sp.…”