When oxygen, produced by cyanobacteria as a by-product of photosynthesis, started appearing in the atmosphere some 2.5 billion years ago, it had profound impact on life on Earth. Anaerobic prokaryotes that came into contact with oxygen had to develop systems for its detoxification, and the oxygen molecule also paved the way for aerobic organisms and influenced the evolution of multi-cellular organisms (see e.g. [1]).

The flavodiiron protein (FDP) family of enzymes is widespread and found in all domains of life. They play key roles during oxidative and nitrosative stress by catalyzing oxygen and/or NO reduction and therefore function in the scavenging and detoxification of these gases (for a review see [2]). Interestingly, some members of the FDP family are bifunctional, reducing both O2 and NO equally well, and some show preferences for either substrate.

The FDPs are soluble enzymes containing a di‑iron active site and a flavin mononucleotide (FMN) cofactor. The FDPs are homodimeric, arranged in an antiparallell fashion such that the two active sites are located at the two monomer-monomer interfaces (see Fig. 1). The name flavodiiron proteins derives from the modular structure of the protein with an N-terminal domain (di‑iron containing) homologous to metallo-β-lactamases, and a C-terminus similar to flavodoxins [2]. The FDPs from Moorella thermoacetica (Mt) and Thermotoga maritima (Tm), the structures of which are shown in Fig. 1, both belong to the class A FDPs, which are widespread among bacteria and archaea. The active di‑iron site is bridged by a hydroxyl group and coordinated by three carboxylates and four histidines. The di‑iron site of one monomer is in close proximity to the FMN cofactor (the closest FMN-diiron site distance is <4 Å) of the other monomer. The two iron ions in the active site are denoted FeD (distal) and FeP (proximal) referring to their distance from the FMN cofactor. The FMN cofactor transfers electrons to the active site and is in turn rereduced by NADH via an NADH:rubredoxin oxidoreductase.

Diiron sites with carboxylate/histidine coordination are present in many enzymes, e.g. alternative oxidases which reduces O2 to H2O in mitochondria, and monooxygenases, such as methane monooxygenase (MMO) which activates O2 in the reduction of methane to methanol [2,7]. Dual capability for O2 and NO reduction is also exhibited by the heme‑copper oxidase superfamily (HCuO), membrane-bound enzymes primarily involved in oxygen respiration and energy conservation (for reviews see [[8], [9], [10], [11]]). In this family, the O2-reducing (and energy-conserving, O2R) members have a binuclear active site with a high-spin heme and a Cu ion, whereas the NO-reducing members (NOR) have replaced the Cu for an Fe. Interestingly, the NORs catalyze also O2-reduction (see e.g. [12,13]) and some O2R families catalyze also NO reduction (see e.g. [14,15]). We previously studied the NO reduction mechanism in FDPs by density functional theory (DFT) calculations on a model derived from the Tm FDP [16]. In this work we have used the same model to study the O2 reduction (Eq. (1)) in FDPs.O2+4H++4e−→2H2O

For NO reduction as catalyzed by the FDPs, there is a reasonable amount of experimental information available, mainly for the Tm FDP [6,[17], [18], [19]]. For the O2 reduction there is much less detailed experimental information available, essentially the only detailed study on the mechanism concerns the Treponema denticola (Td) FDP, for which the formation and decay of a peroxide intermediate has been observed [20]. For several FDPs there is information available for the NO and O2 steady-state activities [3,5,6,18,[20], [21], [22], [23]] and an observation from those is that in most cases the O2 reduction activity is higher than the NO reduction activity, in some cases significantly higher. For example, the Tm FDP shows a steady-state activity (kcat) of 4s−1 for O2, and 0.6s−1 for NO. The corresponding values for Td FDP are 28s−1 for O2, and 1.3s−1 for NO. There are, however, exceptions to this, e.g. the E.coli FDP catalyzes NO reduction at 4s−1 and O2 reduction at <1s−1 [20]. There are also a few mutagenesis studies, both on variations within the active site [6], and in the vicinity of the active site [3,17,24,25], which provide some mechanistic information. However, no clear structural properties have been possible to link to substrate preferences.

The main purpose of the present study is to elucidate the reaction mechanism for O2 reduction in FDP. By comparing the sequences of O2 and NO reduction in the FDPs, we increase the understanding of each of the two mechanisms, and contribute to an explanation of the generally large difference in steady-state activity between the two substrates. We also compare to other O2 reducing enzymes with a bimetallic active site, in particular the HCuO family, which also reduce both O2 and NO, and for which a considerable amount of mechanistic investigations has been performed [[12], [13], [14], [15],26].