Reductive dioxygen scavenging by flavo-diiron proteins of Clostridium acetobutylicum.
Ontology highlight
ABSTRACT:
SUBMITTER: Hillmann F
PROVIDER: S-EPMC2629652 | biostudies-literature | 2009 Jan
REPOSITORIES: biostudies-literature
Publications
Reductive dioxygen scavenging by flavo-diiron proteins of Clostridium acetobutylicum.
Hillmann Falk F Riebe Oliver O Fischer Ralf-Jörg RJ Mot Augustin A Caranto Jonathan D JD Kurtz Donald M DM Bahl Hubert H
FEBS letters 20081211 1
Two flavo-diiron proteins (FDPs), FprA1 and FprA2, are up-regulated when the strictly anaerobic solvent producer, Clostridium acetobutylicum, is exposed to dioxygen. These two FDPs were purified following heterologous overexpression in Escherichia coli as N-terminal Strep-tag fusion proteins. The recombinant FprA1 and FprA2 were found to be homodimeric and homotetrameric, respectively, and both FDPs functioned as terminal components of NADH oxidases (NADH:O(2) oxidoreductases) when using C. acet ...[more]
Similar Datasets
Project description:Flavo-diiron proteins (FDPs) are non-heme iron containing enzymes that are widespread in anaerobic bacteria, archaea, and protozoa, serving as the terminal components to dioxygen and nitric oxide reductive scavenging pathways in these organisms. FDPs contain a dinuclear iron active site similar to that in hemerythrin, ribonucleotide reductase, and methane monooxygenase, all of which can bind NO and O2. However, only FDP competently turns over NO to N2O. Here, EPR and Mössbauer spectroscopies allow electronic characterization of the diferric and diferrous species of FDP. The exchange-coupling constant J (Hex = JS1·S2) was found to increase from +20 cm-1 to +32 cm-1 upon reduction of the diferric to the diferrous species, indicative of (1) at least one hydroxo bridge between the iron ions for both states and (2) a change to the diiron core structure upon reduction. In comparison to characterized diiron proteins and synthetic complexes, the experimental values were consistent with a dihydroxo bridged diferric core, which loses one hydroxo bridge upon reduction. DFT calculations of these structures gave values of J and Mössbauer parameters in agreement with experiment. Although the crystal structure shows a hydrogen bond between the iron bound aspartate and the bridging solvent molecule, the DFT calculations of structures consistent with the crystal structure gave calculated values of J incompatible with the spectroscopic results. We conclude that the crystal structure of the diferric state does not represent the frozen solution structure and that a mono-?-hydroxo diferrous species is the catalytically functional state that reacts with NO and O2. The new EPR spectroscopic probe of the diferric state indicated that the diferric structure of FDP prior to and immediately after turnover with NO are flavin mononucleotide (FMN) dependent, implicating an additional proton transfer role for FMN in turnover of NO.
| S-EPMC5898632 | biostudies-literature
Project description:Flavo-diiron proteins (FDPs) are widespread in anaerobic bacteria, archaea, and protozoa, where they serve as the terminal components of dioxygen and nitric oxide reductive scavenging pathways. FDPs contain an N,O-ligated diiron site adjacent to a flavin mononucleotide (FMN) cofactor. The diiron site is structurally similar to those in hemerythrin, ribonucleotide reductase, and methane monooxygenase. However, only FDPs turn over NO to N2O at significant rates and yields. Previous studies revealed sequential binding of two NO molecules to the diferrous site, forming mono- and dinitrosyl intermediates leading to N2O formation. In the present work, these mono- and dinitrosyl intermediates have been characterized by EPR and Mössbauer spectroscopies and DFT calculations. Our results show that the iron proximal to the cofactor binds the first NO to form the diiron mononitrosyl complex, implying the iron distal to the FMN binds the second NO to form the diiron dinitrosyl intermediate. The exchange-coupling constants, J (H = JS1·S2), were found to differ substantially, +17 cm-1 for the diiron mononitrosyl and +60 cm-1 for the diiron dinitrosyl. Notwithstanding this large difference, our findings indicate retention of at least one hydroxo bridge throughout the NOR catalytic cycle. The Mossbauer hyperfine parameters and DFT calculations confirmed a semibridging NO- ligand in the mononitrosyl intermediate that lowers the exchange parameter. The DFT calculations on the dinitrosyl intermediate suggest a contribution to J from direct exchange between the S = 1 spins on the NO- ligands, which could initiate N-N bond formation. Our results provide insight into why FDPs are the only known nonheme diiron enzymes that competently turn over NO to N2O.
| S-EPMC6602092 | biostudies-literature
Project description:Flavo-diiron proteins (FDPs) contain non-heme diiron and proximal flavin mononucleotide (FMN) active sites and function as terminal components of a nitric oxide reductase (NOR) and/or a four-electron dioxygen reductase (O(2)R). While most FDPs show similar structural, spectroscopic, and redox properties, O(2)R and NOR activities vary significantly among FDPs. A potential source of this variability is the iron ligation status of a conserved His residue that provides an iron ligand in all known FDP structures but one, where this His residue is rotated away from iron and replaced by a solvent ligand. In order to test the effect of this His ligation status, we changed this ligating His residue (H90) in Thermotoga maritima (Tm) FDP to either Asn or Ala. The wild-type Tm FDP shows significantly higher O(2)R than NOR activity. Single crystal X-ray crystallography revealed a remarkably conserved diiron site structure in the H90N and -A variants, differing mainly by either Asn or solvent coordination, respectively, in place of H90. The steady-state activities were minimally affected by the H90 substitutions, remaining significantly higher for O(2)R versus NOR. The pre-steady-state kinetics of the fully reduced FDP with O(2) were also minimally affected by the H90 substitutions. The results indicate that the coordination status of this His ligand does not significantly modulate the O(2)R or NOR activities, and that FDPs can retain these activities when the individual iron centers are differentiated by His ligand substitution. This differentiation may have implications for the O(2)R and NOR mechanisms of FDPs.
| S-EPMC3508297 | biostudies-literature
Project description:A growing subset of metalloenzymes activates dioxygen with nonheme diiron active sites to effect substrate oxidations that range from the hydroxylation of methane and the desaturation of fatty acids to the deformylation of fatty aldehydes to produce alkanes and the six-electron oxidation of aminoarenes to nitroarenes in the biosynthesis of antibiotics. A common feature of their reaction mechanisms is the formation of O2 adducts that evolve into more reactive derivatives such as diiron(II,III)-superoxo, diiron(III)-peroxo, diiron(III,IV)-oxo, and diiron(IV)-oxo species, which carry out particular substrate oxidation tasks. In this review, we survey the various enzymes belonging to this unique subset and the mechanisms by which substrate oxidation is carried out. We examine the nature of the reactive intermediates, as revealed by X-ray crystallography and the application of various spectroscopic methods and their associated reactivity. We also discuss the structural and electronic properties of the model complexes that have been found to mimic salient aspects of these enzyme active sites. Much has been learned in the past 25 years, but key questions remain to be answered.
| S-EPMC5920527 | biostudies-literature
Project description:Background:Biofilms are cell communities wherein cells are embedded in a self-produced extracellular polymeric substances (EPS). The biofilm of Clostridium acetobutylicum confers the cells superior phenotypes and has been extensively exploited to produce a variety of liquid biofuels and bulk chemicals. However, little has been known about the physiology of C. acetobutylicum in biofilm as well as the composition and biosynthesis of the EPS. Thus, this study is focused on revealing the cell physiology and EPS composition of C. acetobutylicum biofilm. Results:Here, we revealed a novel lifestyle of C. acetobutylicum in biofilm: elimination of sporulation and vegetative growth. Extracellular polymeric substances and wire-like structures were also observed in the biofilm. Furthermore, for the first time, the biofilm polysaccharides and proteins were isolated and characterized. The biofilm contained three heteropolysaccharides. The major fraction consisted of predominantly glucose, mannose and aminoglucose. Also, a great variety of proteins including many non-classically secreted proteins moonlighting as adhesins were found considerably present in the biofilm, with GroEL, a S-layer protein and rubrerythrin being the most abundant ones. Conclusions:This study evidenced that vegetative C. acetobutylicum cells rather than commonly assumed spore-forming cells were essentially the solvent-forming cells. The abundant non-classically secreted moonlighting proteins might be important for the biofilm formation. This study provides the first physiological and molecular insights into C. acetobutylicum biofilm which should be valuable for understanding and development of the biofilm-based processes.
| S-EPMC6245871 | biostudies-literature
Project description:Flavo-diiron proteins (FDPs) function as anaerobic nitric oxide scavengers in some microorganisms, catalyzing reduction of nitric to nitrous oxide. The FDP from Thermotoga maritima can be prepared in a deflavinated form with an intact diferric site (deflavo-FDP). Hayashi et al. [(2010) Biochemistry 49, 7040-7049] reported that reaction of NO with reduced deflavo-FDP produced substoichiometric N2O. Here we report a multispectroscopic approach to identify the iron species in the reactions of deflavo-FDP with NO. Mössbauer spectroscopy identified two distinct ferrous species after reduction of the antiferromagnetically coupled diferric site. Approximately 60% of the total ferrous iron was assigned to a diferrous species associated with the N2O-generating pathway. This pathway proceeds through successive diferrous-mononitrosyl (S = (1)/2 Fe(II){FeNO}(7)) and diferrous-dinitrosyl (S = 0 [{FeNO}(7)]2) species that form within ?100 ms of mixing of the reduced protein with NO. The diferrous-dinitrosyl intermediate converted to an antiferromagnetically coupled diferric species that was spectroscopically indistinguishable from that in the starting deflavinated protein. These diiron species closely resembled those reported for the flavinated FDP [Caranto et al. (2014) J. Am. Chem. Soc. 136, 7981-7992], and the time scales of their formation and decay were consistent with the steady state turnover of the flavinated protein. The remaining ?40% of ferrous iron was inactive in N2O generation but reversibly bound NO to give an S = (3)/2 {FeNO}(7) species. The results demonstrate that N2O formation in FDPs can occur via conversion of S = 0 [{FeNO}(7)]2 to a diferric form without participation of the flavin cofactor.
| S-EPMC4159209 | biostudies-literature
Project description:One of the biggest limitations in the study and engineering of anaerobic Clostridium organisms is the lack of strong fluorescent reporters capable of strong and real-time fluorescence. Recently, we developed a strong fluorescent reporter system for Clostridium organisms based on the FAST protein. Here, we report the development of two new strong fluorescent reporter systems for Clostridium organisms based on the HaloTag and SNAP-tag proteins, which produce strong fluorescent signals when covalently bound to fluorogenic ligands. These new fluorescent reporters are orthogonal to the FAST ligands and to each other, allowing for simultaneous labeling and visualization. We used HaloTag and SNAP-tag to label the strictly anaerobic organisms Clostridium acetobutylicum and Clostridium ljungdahlii We have also identified a new strong promoter for protein expression in C. acetobutylicum, based on the phosphotransacetylase gene (pta) from C. ljungdahlii Furthermore, the HaloTag and the SNAP-tag, in combination with the previously described FAST system, were successfully used to measure cell populations in bacterial mixed cultures and showed the simultaneous orthogonal labeling of HaloTag and SNAP-tag together with the FAST protein reporter. Finally, we show the expression of recombinant fusion protein of FAST and the ZapA division protein (from C. acetobutylicum) in C. ljungdahlii. The availability of multiple strong fluorescent reporters is a major addition to the genetic toolkit of Clostridium and other anaerobes that will lead to better understanding of these unique organisms.IMPORTANCE Up to this point, assays and methods involving fluorescent reporter proteins were unavailable or limited in Clostridium organisms and other strict anaerobes. Green fluorescent protein (GFP), mCherry, and flavin-binding proteins (and their derivatives) have been used only in a few clostridia with limited success and yielded low fluorescence compared to aerobic microbial systems. Recently, we reported a new strong fluorescent reporter system based on the FAST protein as a first step in expanding the fluorescence-based reporters for Clostridium and other anaerobic microbial platforms. Additional strong orthogonal fluorescent proteins, with distinct emission spectra are needed to allow for (i) multispecies tracking within the growing field of microbial cocultures and microbiomes, (ii) protein localization and tracking in anaerobes, and (iii) identification and development of natural and synthetic promoters, ribosome-binding sites (RBS), and terminators for optimal protein expression in anaerobes. Here, we present two new strong fluorescent reporter systems based on the HaloTag and SNAP-tag proteins.
| S-EPMC7531948 | biostudies-literature
Project description:Prokaryotic genes are frequently organized in multicistronic operons (or transcriptional units, TUs), and usually the regulatory motifs for the whole TU are located upstream of the first TU gene. Although the number of sequenced genomes has increased dramatically, experimental information on TU organization is extremely limited. Even for organisms as extensively studied as Escherichia coli and Bacillus subtilis, TU annotation is far from complete. It therefore becomes imperative to rely on computational approaches to complement experimental information. Here we present a TU map for the obligate anaerobe Clostridium acetobutylicum ATCC 824. This map is largely based on the distance between pairs of consecutive genes but enhanced and refined by predictions of several types of promoters (sigmaA, sigmaE and sigmaF/G) and rho-independent terminator structures. Based on the set of known C.acetobutylicum TUs, the presented TU map offers an 88% prediction accuracy.
| S-EPMC390361 | biostudies-literature
Project description:The unique active site of flavo-diiron proteins (FDPs) consists of a nonheme diiron-carboxylate site proximal to a flavin mononucleotide (FMN) cofactor. FDPs serve as the terminal components for reductive scavenging of dioxygen or nitric oxide to combat oxidative or nitrosative stress in bacteria, archaea, and some protozoan parasites. Nitric oxide is reduced to nitrous oxide by the four-electron reduced (FMNH2-Fe(II)Fe(II)) active site. In order to clarify the nitric oxide reductase mechanism, we undertook a multispectroscopic presteady-state investigation, including the first Mössbauer spectroscopic characterization of diiron redox intermediates in FDPs. A new transient intermediate was detected and determined to be an antiferromagnetically coupled diferrous-dinitrosyl (S = 0, [{FeNO}(7)]2) species. This species has an exchange energy, J ? 40 cm(-1) (JS1 ° S2), which is consistent with a hydroxo or oxo bridge between the two irons. The results show that the nitric oxide reductase reaction proceeds through successive formation of diferrous-mononitrosyl (S = ½, Fe(II){FeNO}(7)) and the S = 0 diferrous-dinitrosyl species. In the rate-determining process, the diferrous-dinitrosyl converts to diferric (Fe(III)Fe(III)) and by inference N2O. The proximal FMNH2 then rapidly rereduces the diferric site to diferrous (Fe(II)Fe(II)), which can undergo a second 2NO ? N2O turnover. This pathway is consistent with previous results on the same deflavinated and flavinated FDP, which detected N2O as a product (Hayashi Biochemistry 2010, 49, 7040). Our results do not support other proposed mechanisms, which proceed either via "super-reduction" of [{FeNO}(7)]2 by FMNH2 or through Fe(II){FeNO}(7) directly to a diferric-hyponitrite intermediate. The results indicate that an S = 0 [{FeNO}(7)}]2 complex is a proximal precursor to N-N bond formation and N-O bond cleavage to give N2O and that this conversion can occur without redox participation of the FMN cofactor.
| S-EPMC4063189 | biostudies-literature
Project description:For numerous enzymes reactive toward small gaseous compounds, growing evidence indicates that these substrates diffuse into active site pockets through defined pathways in the protein matrix. Toluene/o-xylene monooxygenase hydroxylase is a dioxygen-activating enzyme. Structural analysis suggests two possible pathways for dioxygen access through the α-subunit to the diiron center: a channel or a series of hydrophobic cavities. To distinguish which is utilized as the O(2) migration pathway, the dimensions of the cavities and the channel were independently varied by site-directed mutagenesis and confirmed by X-ray crystallography. The rate constants for dioxygen access to the diiron center were derived from the formation rates of a peroxodiiron(III) intermediate, generated upon treatment of the diiron(II) enzyme with O(2). This reaction depends on the concentration of dioxygen to the first order. Altering the dimensions of the cavities, but not the channel, changed the rate of dioxygen reactivity with the enzyme. These results strongly suggest that voids comprising the cavities in toluene/o-xylene monooxygenase hydroxylase are not artifacts of protein packing/folding, but rather programmed routes for dioxygen migration through the protein matrix. Because the cavities are not fully connected into the diiron active center in the enzyme resting state, conformational changes will be required to facilitate dioxygen access to the diiron center. We propose that such temporary opening and closing of the cavities may occur in all bacterial multicomponent monooxygenases to control O(2) consumption for efficient catalysis. Our findings suggest that other gas-utilizing enzymes may employ similar structural features to effect substrate passage through a protein matrix.
| S-EPMC3169157 | biostudies-literature