On 2016 Feb 05, James Murray commented:

The superoxide dependent nitrogenase described in this (Ribbe M, 1997) paper is extremely unlikely to exist.

The paper describes the purification of the components of an oxygen-tolerant nitrogenase, not homologous to the known nif,vnf, or anf-type, from Streptomyces thermoautotrophicus UBT1, a thermophilic carboxydotroph.

Results published in February 2016 (MacKellar D, 2016) show that three independent isolates of S. thermoautotrophicus, including the original UBT1 strain, do not grow in the absence of combined nitrogen and are incapable of incorporating isotopically labelled dinitrogen into biomass, nor do they contain the claimed superoxide dependent nitrogenase genes. The N-terminal sequences assigned to nitrogenase components in Ribbe M, 1997, and the full DNA gene sequences in the PhD thesis of Carla Hofmann-Findeklee (2000, KF951061.1, KF951060.1, KF951059.1, KF956113.1) are found at near-identity in Bacillus schlegelii DSM9132 (recently renamed to Hydrogenibacillus schlegelii, "SdnMSL-like" sequences in KT861421.1), a non-diazotrophic thermophilic carboxydotrophic organism isolated in the Meyer lab (Krüger & Meyer, 1984) and known to be cultured in the Meyer laboratory in 1994 (Hänzelmann, 1994). The independently isolated B. schlegelii DSM2000 strain also has these sequences at near-identity. The closest relatives to these sequences are to Firmicutes and not Actinomycetes like S. thermoautotrophiucs. The four "nitrogenase" sequences are easily identified as encoding a superoxide dismutase ("st2", sdnO), and a three-subunit aerobic carbon monoxide dehydrogenase ("st1", sdnMSL).

Ribbe M, 1997 relies on an ammonia production assay to determine the nitrogenase activity. This assay is known to have a high background due to environmental ammonia and protein deamination. Incorporation of isotopically labelled dinitrogen is usually considered the gold standard for the identification of a nitrogenase enzyme. No incorporation of isotopically labelled nitrogen into ammonia is shown using the claimed biochemical nitrogenase preparation. The cells were grown in media with 1.5 g/l ammonium chloride, so there was no selection for diazotrophy. No published demonstration of the superoxide dependent nitrogenase has occurred outside the Meyer laboratory.

The nitrogenase scheme described in Ribbe M, 1997 is chemically and biologically implausible. There is no known ATPase domain, as required by the proposed reaction scheme, in any of the described proteins. The known nitrogenase types require the highly reducing ferredoxin or flavodoxin as reductants. Superoxide is an unlikely electron donor for a nitrogenase, as it is not as reducing as even NADPH or NADH, and is reactive and toxic. No other biologically productive use of superoxide as an electron donor is known. An aerobic reduction of nitrogen to ammonia is unknown, and unlikely, as under the highly reducing conditions, oxygen would most probably be reduced in preference to nitrogen. The rate of activity described is too low to be that of a biological enzyme supporting diazotrophic growth, as it would take the proposed nitrogenase over 100 hours just to replace the nitrogen in the enzyme itself, which is also incompatible with the claimed rate of diazotrophic growth of S. thermoautotrophicus (Gadkari D, 1992).

To summarize:

• Recent evidence suggests that three independently isolated strains of S. thermoautotrophicus are not diazotrophic.

• If the Meyer laboratory did contaminate their S. thermoautotrophicus culture with a strain of B. schlegelii (such as the DSM9132 strain), we would observe the N-terminal sequences presented here and the DNA gene sequences also produced in the Meyer laboratory.

• The extremely low activity "nitrogenase" was described based on a problematic ammonia production assay.

• A superoxide-dependent aerobic nitrogenase is chemically and biologically implausible.

Declaration: I am an author on the MacKellar D, 2016 paper, but this comment is entirely my own.

References:

Bernd Krüger and Ortwin Meyer. Thermophilic bacilli growing with carbon monoxide. Archives of Microbiology, 139(4):402–408, 1984.

Petra Hänzelmann. Isolierung und Charakterisierung von Kohlenmonoxid-Dehydrogenase aus dem obligat thermophilen Bakterium Bacillus schlegelii. Diplomarbeit thesis, University of Bayreuth, 1994.

Carla Hofmann-Findeklee. Molekularbiologische Untersuchung der Strukturgene des aeroben N2-fixierenden Systems von Streptomyces thermoautotrophicus sowie funktionelle Charakterisierung von rekombinantem SdnO. PhD thesis, University of Bayreuth, 2000.

On 2016 Feb 05, James Murray commented:

The superoxide dependent nitrogenase described in this (Ribbe M, 1997) paper is extremely unlikely to exist.

The paper describes the purification of the components of an oxygen-tolerant nitrogenase, not homologous to the known nif,vnf, or anf-type, from Streptomyces thermoautotrophicus UBT1, a thermophilic carboxydotroph.

Results published in February 2016 (MacKellar D, 2016) show that three independent isolates of S. thermoautotrophicus, including the original UBT1 strain, do not grow in the absence of combined nitrogen and are incapable of incorporating isotopically labelled dinitrogen into biomass, nor do they contain the claimed superoxide dependent nitrogenase genes. The N-terminal sequences assigned to nitrogenase components in Ribbe M, 1997, and the full DNA gene sequences in the PhD thesis of Carla Hofmann-Findeklee (2000, KF951061.1, KF951060.1, KF951059.1, KF956113.1) are found at near-identity in Bacillus schlegelii DSM9132 (recently renamed to Hydrogenibacillus schlegelii, "SdnMSL-like" sequences in KT861421.1), a non-diazotrophic thermophilic carboxydotrophic organism isolated in the Meyer lab (Krüger & Meyer, 1984) and known to be cultured in the Meyer laboratory in 1994 (Hänzelmann, 1994). The independently isolated B. schlegelii DSM2000 strain also has these sequences at near-identity. The closest relatives to these sequences are to Firmicutes and not Actinomycetes like S. thermoautotrophiucs. The four "nitrogenase" sequences are easily identified as encoding a superoxide dismutase ("st2", sdnO), and a three-subunit aerobic carbon monoxide dehydrogenase ("st1", sdnMSL).

Ribbe M, 1997 relies on an ammonia production assay to determine the nitrogenase activity. This assay is known to have a high background due to environmental ammonia and protein deamination. Incorporation of isotopically labelled dinitrogen is usually considered the gold standard for the identification of a nitrogenase enzyme. No incorporation of isotopically labelled nitrogen into ammonia is shown using the claimed biochemical nitrogenase preparation. The cells were grown in media with 1.5 g/l ammonium chloride, so there was no selection for diazotrophy. No published demonstration of the superoxide dependent nitrogenase has occurred outside the Meyer laboratory.

The nitrogenase scheme described in Ribbe M, 1997 is chemically and biologically implausible. There is no known ATPase domain, as required by the proposed reaction scheme, in any of the described proteins. The known nitrogenase types require the highly reducing ferredoxin or flavodoxin as reductants. Superoxide is an unlikely electron donor for a nitrogenase, as it is not as reducing as even NADPH or NADH, and is reactive and toxic. No other biologically productive use of superoxide as an electron donor is known. An aerobic reduction of nitrogen to ammonia is unknown, and unlikely, as under the highly reducing conditions, oxygen would most probably be reduced in preference to nitrogen. The rate of activity described is too low to be that of a biological enzyme supporting diazotrophic growth, as it would take the proposed nitrogenase over 100 hours just to replace the nitrogen in the enzyme itself, which is also incompatible with the claimed rate of diazotrophic growth of S. thermoautotrophicus (Gadkari D, 1992).

To summarize:

• Recent evidence suggests that three independently isolated strains of S. thermoautotrophicus are not diazotrophic.

• If the Meyer laboratory did contaminate their S. thermoautotrophicus culture with a strain of B. schlegelii (such as the DSM9132 strain), we would observe the N-terminal sequences presented here and the DNA gene sequences also produced in the Meyer laboratory.

• The extremely low activity "nitrogenase" was described based on a problematic ammonia production assay.

• A superoxide-dependent aerobic nitrogenase is chemically and biologically implausible.

Declaration: I am an author on the MacKellar D, 2016 paper, but this comment is entirely my own.

References:

Bernd Krüger and Ortwin Meyer. Thermophilic bacilli growing with carbon monoxide. Archives of Microbiology, 139(4):402–408, 1984.

Petra Hänzelmann. Isolierung und Charakterisierung von Kohlenmonoxid-Dehydrogenase aus dem obligat thermophilen Bakterium Bacillus schlegelii. Diplomarbeit thesis, University of Bayreuth, 1994.

Carla Hofmann-Findeklee. Molekularbiologische Untersuchung der Strukturgene des aeroben N2-fixierenden Systems von Streptomyces thermoautotrophicus sowie funktionelle Charakterisierung von rekombinantem SdnO. PhD thesis, University of Bayreuth, 2000.