Flavodiiron proteins
Transition from an anaerobic to an aerobic world
Life originated on Earth ca 3.5 billion years ago in an anoxic and reducing environment that sustained different types of anaerobic metabolisms. Molecular oxygen, if present, would be vestigial and confined to restricted niches. This situation changed dramatically upon appearance of oxygenic photosynthesis performed by cyanobacteria, leading several million years after to what has been called the Great Oxidation Event, about 2.5 billion years ago. Oxygen in the atmosphere eventually stabilized at around 21%, i.e., a quite oxidizing environment was established, leading to major changes in the cellular metabolism. Existing life forms adopted different strategies to cope with this new challenge: i) avoiding it by inhabiting anaerobic niches, but “evolving” (possibly using already existing cellular components) ways to protect themselves while keeping the intracellular reducing medium, or ii) profiting from it, both for biosynthetic processes and for aerobic respiration. In fact, the highly positive oxygen reduction potential (+0.82 V at standard atmospheric pressure and temperature) allowed extracting maximum energy from cellular food stuff (e.g. glucose), which favoured the evolution of the complex multicellular life forms now present on Earth. However, oxygen also poses a threat to either anaerobic or aerobic living forms (the ‘dark side’ of oxygen): although the oxygen reactivity is limited by its spin triplet ground state, it may still react fairly rapidly with radical organic compounds, such as flavins or quinones, or with metals, such as the widespread iron centres. Moreover, upon successive one electron reduction steps, oxygen forms highly reactive species (ROS), the superoxide anion, hydrogen peroxide and the hydroxyl radical, that may further and rapidly react with multiple cellular components (again, metal centres, thiols, lipids and nucleotides). As a result, all cellular forms analysed to date contain enzymatic systems to sense, use, and eliminate molecular oxygen and the derived reactive species. Molecular oxygen can be fully reduced to water by respiratory membrane-bound oxygen reductases (haem-copper reductases, alternative oxidase (AOX) and cytochrome bd) and O2-reducing non-haem diiron enzymes, or by the flavodiiron proteins (FDPs), the more recently discovered family of oxygen reductases. Some of these enzymes are also endowed with nitric oxide (NO) reductase activity.
Flavodiiron proteins - a short historical account
The first reported flavodiiron protein, and for which a function was promptly proposed, was isolated in 1993 from the then considered strict anaerobic bacterium Desulfovibrio (D.) gigas. D. gigas FDP was initially named rubredoxin:oxygen oxidoreductase (ROO), as it coupled the oxidation of rubredoxin (reduced by an NADH oxidoreductase) to oxygen reduction to water. Based on amino acid sequence analysis, Wasserfallen and co-workers identified a new family of flavo(metallo)enzymes, then named A-type flavoproteins (FprA), and did a basic characterization of Escherichia coli and Synechocystis FDPs, already proposing at the time a classification based on the different cofactors (domains). A landmark in FDP history was the determination of the crystallographic structure of D. gigas FDP in 2000, which revealed for the first time the existence of the β-lactamase-like fold harbouring a non-haem diiron centre. Whereas most focus was given to the oxygen detoxification role, a striking observation concerned the proposal by Gardner and co-workers that E. coli FDP is an NO reductase, soon after confirmed by us through amperometric measurements with the isolated enzyme. In 2003, Kurtz and co-workers reported the first example of a bi-functional FDP (from Moorella thermoacetica) endowed with both NO and O2 reductase activity, and coined the term “Flavodiiron protein” for this enzyme family. In the meantime, a significant line of research in the FDP field developed, addressing the involvement of cyanobacterial FDPs in photoprotection of oxygenic photosynthesis. Over the last ten years, other bifunctional FDPs were characterized. as well as FDPs more selective either for NO (E. coli and Salmonella enterica FDPs) or for oxygen (e.g., eukaryotic FDPs from anaerobic protozoa or from methanogens)
Presently, FDPs are recognized as a large family of enzymes, widespread in all life Domains (Bacteria, Archaea and Eukarya), with a key function in oxygen and/or nitric oxide detoxification.
The FDP family and their modular nature
The FDP minimal structural unit is composed by two domains: the N-terminus is homologous to metallo-β-lactamases and the C-terminus is similar to small-flavodoxins These two domains constitute the common denominator of this enzyme family, and gave rise to the general term Flavodiiron Proteins (FDPs).
This minimal common denominator is present in the Class A FDPs, so far the most widespread among prokaryotes. Yet, more complex and interesting modular structures have been studied or encountered in the genomes, with extra domains at the C-terminal part of the protein: rubredoxins (Class B FDPs, so-called flavorubredoxins, so far only found in the Proteobacteria phylum, particularly in Enterobacteriales), flavin reductases (Class C FDPs, in all cyanobacteria whose genomes have been so far sequenced, as well as in several photosynthetic eukaryotes), and even more complex arrangements, involving fusion with short-spaced rubredoxins, NADH:rubredoxin oxidoreductases and NAD(P)H:flavin oxidoreductases.
Another interesting feature of FDPs concerns the electron transfer chains supplying electrons to FDPs to act as O2/NO reductases. NAD(P)H appears to be the common primary electron source, the sole exception being the F420 cofactor in methanogenic Archaea, which directly reduces the Methanobrevibacter arboriphilus and Methanothermobacter thermoautotrophicum FDPs. A common feature of several FDPs is the ability to oxidize rubredoxins, small electron transfer proteins with a [FeCys4] centre, which are reduced by flavin-containing NAD(P)H oxidoreductases.
Class B FDPs have the rubredoxin fused to the C-terminus as an extra domain, bypassing the need for an external rubredoxin partner. Similarly, Class C FDPs have an extra C-terminal flavin reductase-like domain that directly accepts electrons from NAD(P)H, thus condensing the electron transfer chain into a single polypeptide. It should be stressed that in many instances the physiological electron donor to the FDPs remains unknown, particularly in organisms whose genomes lack genes encoding rubredoxins.
The structure of flavodiiron proteins
The flavodiiron proteins are isolated as homodimers or homotetramers (dimer of dimers), and their crystallographic structures confirm this quaternary arrangement. In Class A FDPs each 40-48 kDa monomer is constituted by two domains: the N-terminal domain shows an aββa topology characteristic of metallo-β-lactamase-like folds, harbouring a diiron centre; and the C-terminal domain with an aβa topology similar to short- chain flavodoxins that binds non-covalently a flavin mononucleotide (FMN). The distance between the diiron centre and the FMN within the same monomer is ca. 40 Å, precluding direct electron transfer. However, the minimal functional unit in structurally characterized FDPs is a dimer arranged in a head-to-tail orientation, in which the diiron centre from one subunit is at ca. 4 Å from the FMN from the other monomer, thus ensuring an efficient electron transfer between the two centres.
The diiron centre of flavodiiron proteins
The binuclear site of FDPs is located within a groove at the interface between the two inner β-sheets of the β-lactamase-like fold. It is surrounded by aβ loops and β-hairpins, being in the vicinity and confined by the C-terminal domain of the opposing monomer. Differently from other metallo-β-lactamase-like proteins, the metal site is covered by a unique two-stranded β-sheet that, together with the dimer mate, hinders the access of large substrates, such as β-lactams.
Available FDP´s crystal structures present almost strictly conserved amino acid metal ligands. The diiron centre is coordinated by a µ-(hydr)oxo species, three carboxylate and four imidazole ligands, from a highly conserved motif, H81-x-E83-x-D85-H86-x61-H148-X18-D167-x60-H228 . The iron atom closest to the FMN (proximal iron, FeP) is coordinated by His81, Glu83 and His148, while the distal atom is bound to Asp85, His86 and His228. In D. gigas FDP a water molecule substitutes the His86 imidazole, which adopts a unique chi1 value allowing the establishment of an additional hydrogen bonding network involving Asp225, Asp49 and Gln78 (D. gigas FDP numbering). Both irons are further bridged by the carboxylate group of Asp167 and by the µ-(hydr)oxo species. The two metal sites are penta-coordinated with distorted square pyramidal geometries, with the bases of the pyramids approximately parallel to each other.
Most Class C enzymes may have a distinct iron coordination, as some ligands are not conserved being eventually substituted by non-canonical ligands, such as arginines and glutamines. They show a large variability in the combination of these amino acids in the positions for possible iron binding, which led to the classification of 15 different subtypes of Class C enzymes.
FDPs and other diiron proteins
There is a wealth of diiron proteins displaying the most diverse functions, such as monooxygenases, ribonucleotide reductases, oxygen transporters (haemrythrins) or reducers (alternative oxidases), and ferroxidases (ferritins and haemferritins). Despite containing diiron centres of the histidine/carboxylate family like those of FDPs, all those referred proteins contain their metal site embedded in a four-helix bundle fold, totally dissimilar to that of FDPs (metallo-β-lactamase-like fold). Although the number of histidine and carboxylate ligands also varies between these different proteins, both FDPs and the mentioned four-helix-bundle diiron proteins have in common that they activate/react with oxygen, be it for its transport (haemerythrins), to oxidize ferrous iron (ferritin) or hydrocarbons (e.g. methane monooxygenase), or to generate tyrosyl radicals (ribonucleotide reductases). Only alternative oxidases have as their physiological function the direct reduction of oxygen to water.
O2 versus NO reduction
A major question in the field of the FDPs is whether these are oxygen or nitric oxide reductases, or both. In fact, for the few FDPs so far biochemically characterized, it appears that the three possibilities exist, although one should stress that comprehensive enzymatic studies leading to the determination of key kinetic parameters are scarce. In terms of electron donating capabilities, the fully electron loaded FDP (considering only the minimal core domain) has four electrons available (two in the FMN, two in the diiron centre) for the reduction of the oxygen molecule to water or for the reduction of two NO molecules to N2O. An interesting outlier concerns the E. coli FDP, flavorubredoxin, whose one-electron reduced FMN (semiquinone) is kinetically stable. However, the extra rubredoxin domain may still provide the fourth reducing equivalent.
Physiological functions of flavodiiron proteins
Most prokaryotic FDPs are generally considered to be cytoplasmic enzymes, due to the lack of signal peptides in their sequences. However, in cyanobacteria, which contain multiple copies of FDPs, some of them are proposed to be membrane-associated under certain conditions, namely close to photosystem II. In eukrayotes, FDPs may be located in organelles: for example, in the unicellular protozoan Trichomonas vaginalis, one of the encoded FDPs is located in hydrogenosomes, which are organelles remnant of mitochondria but metabolically adapted to anoxic life; in the algae Chlamydomonas reinnhardtii, as probably in all eukaryotic oxygenic phototrophs that contain FDPs, they are located in the chloroplasts.
Accordingly to the first function proposed for FDPs, as an oxygen reductase, the expression of FDPs is up-regulated by exposure of several anaerobic microbes to low oxygen levels. The work of E.-M. Aro and co-workers, within the FDP field concerns the role of cyanobacterial FDPs in protection of oxygenic photosynthesis, particularly by participating in oxygen photoreduction and protecting photosystems I and II under variable conditions, such as light intensity and CO2 availability. It is not a surprise that cyanobacteria, which produce O2 as a byproduct of their photosynthetic metabolism, are particularly rich in FDPs, which afford a direct protection against oxygen. An important role for FDPs was also shown for protection of the highly oxygen sensitive enzyme nitrogenase against oxygen, in Anabaena heterocysts.
In the enterobacteria E. coli and Salmonella, FDPs act as NO reductases, their expression being significantly up-regulated in cell cultures exposed to authentic NO solutions or NO releasers under anaerobic conditions. For sulfate-reducing bacteria it was established that FDPs afford protection against NO-derived stress under anaerobiosis, through the analysis of the phenotypes of null mutants of the FDP-encoding gene.
The functions of FDPs may be particularly important in the context of host-microbe interactions, both from a host-pathogen strife viewpoint, or simply for survival of human microbiota within a challenging environment. For example, whereas the gut is generally considered an anaerobic milieu, oxygen concentrations can reach up to ~60 μM in the intestinal tract, particularly the colon. On the other hand, NO is generated in the gut as a by-product of denitrification carried out by gut microbiota members and by acidification of nitrite. Therefore, the gut microbial population clearly benefits from O2 and NO detoxification systems such as FDPs. Regarding the immune system weapons against invading microbes, mammalian macrophages/neutrophiles attack pathogens initially through an intense oxidative burst, which is followed by the release of nitric oxide. Therefore, the resistance mechanisms that invading pathogens are endowed with are often considered virulence factors, since they constitute the first line of survival in the host’s hostile environment. Concerning the enterobacterial NO-reducing FDPs, it has been demonstrated that FDPs have an important role in counteracting the nitrosative stress imposed by the host immune system.
