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Reductive dioxygen scavenging by flavo-diiron proteins of Clostridium acetobutylicum.

ABSTRACT: 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. acetobutylicum NADH:rubredoxin oxidoreductase (NROR) and rubredoxin (Rd) as electron transport intermediaries. Both FDPs catalyzed the four-electron reduction of molecular oxygen to water with similar specific activities. The results are consistent with these FDPs functioning as efficient scavengers of intracellular dioxygen under aerobic or microoxic growth conditions.

SUBMITTER: Hillmann F

PROVIDER: S-EPMC2629652 | biostudies-literature | 2009 Jan

REPOSITORIES: biostudies-literature

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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]

PMID: 19084524

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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.

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.

Dataset Information

Reductive dioxygen scavenging by flavo-diiron proteins of Clostridium acetobutylicum.

Publications

Reductive dioxygen scavenging by flavo-diiron proteins of Clostridium acetobutylicum.

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