Detection of the <i>in vivo</i> incorporation of a metal cluster into a protein

Gollan, Ute; Schneider, Klaus; Müller, Achim; Schüddekopf, Kerstin; Klipp, Werner

doi:10.1111/j.1432-1033.1993.tb18003.x

Cited by 54 publications

(44 citation statements)

References 34 publications

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“…However, when biosynthesis of FeMo-co was enabled by addition of nifENX to the minimal Anf system, formation of ethane as a product of acetylene reduction was significantly enhanced in the presence of Mo. This property has been previously characterized in both A. vinelandii and R. capsulatus as the "molybdenum effect," in which FeMo-co can be inserted into the AnfDGK apoprotein instead of FeFe-co (32,(43)(44)(45). The increased formation of ethane under these conditions is thought to arise from the altered polypeptide environment surrounding the cofactor.…”

Section: Discussionmentioning

confidence: 95%

Reconstruction and minimal gene requirements for the alternative iron-only nitrogenase in Escherichia coli

Yang

Xie

Wang

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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All diazotrophic organisms sequenced to date encode a molybdenum-dependent nitrogenase, but some also have alternative nitrogenases that are dependent on either vanadium (VFe) or iron only (FeFe) for activity. In Azotobacter vinelandii, expression of the three different types of nitrogenase is regulated in response to metal availability. The majority of genes required for nitrogen fixation in this organism are encoded in the nitrogen fixation (nif) gene clusters, whereas genes specific for vanadium-or irondependent diazotophy are encoded by the vanadium nitrogen fixation (vnf) and alternative nitrogen fixation (anf) genes, respectively. Due to the complexities of metal-dependent regulation and gene redundancy in A. vinelandii, it has been difficult to determine the precise genetic requirements for alternative nitrogen fixation. In this study, we have used Escherichia coli as a chassis to build an artificial iron-only (Anf) nitrogenase system composed of defined anf and nif genes. Using this system, we demonstrate that the pathway for biosynthesis of the iron-only cofactor (FeFe-co) is likely to be simpler than the pathway for biosynthesis of the molybdenum-dependent cofactor (FeMo-co) equivalent. A number of genes considered to be essential for nitrogen fixation by FeFe nitrogenase, including nifM, vnfEN, and anfOR, are not required for the artificial Anf system in E. coli. This finding has enabled us to engineer a minimal FeFe nitrogenase system comprising the structural anfHDGK genes and the nifBUSV genes required for metallocluster biosynthesis, with nifF and nifJ providing electron transport to the alternative nitrogenase. This minimal Anf system has potential implications for engineering diazotrophy in eukaryotes, particularly in compartments (e.g., organelles) where molybdenum may be limiting.

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Section: Discussionmentioning

confidence: 95%

Reconstruction and minimal gene requirements for the alternative iron-only nitrogenase in Escherichia coli

Yang

Xie

Wang

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…The absence or the almost complete absence of Mo in the growth medium is certainly the most fundamental prerequisite for the derepression of the Moindependent Fe nitrogenase in R. cupsulatus [ 10,21,241. However, there are several further growth factors that also significantly influence the expression of Fe nitrogenase activity.…”

Section: Resultsmentioning

confidence: 99%

“…Anaerobic harvesting of the cells was carried out as described by Gollan et al [21]. The cells, which were about 100-fold concentrated by centrifugation (corresponding to -50 mg protein/ml cell suspension), were disrupted exclusively by lysozyme treatment.…”

Section: Methodsmentioning

confidence: 99%

“…By EPR spectroscopic data and the use of R. cupsulatus mutants, which were defective in the FeMo cofactor synthesis, we were able to demonstrate that the Mo effect is based on the formation of a hybrid enzyme consisting of the Fe nitrogenase apoprotein and the FeMo cofactor of the conventional nitrogenase. Hybrid enzyme formation was shown to be mainly a result of a cluster exchange mechanism [21].…”

Section: I]mentioning

confidence: 99%

“…The Mo effect (increase of the production of C,H, from C,H, after addition of MOO:-to a growing AnifHDK culture) [21] appears to be a specific feature of the iron-only nitrogenases. By EPR spectroscopic data and the use of R. cupsulatus mutants, which were defective in the FeMo cofactor synthesis, we were able to demonstrate that the Mo effect is based on the formation of a hybrid enzyme consisting of the Fe nitrogenase apoprotein and the FeMo cofactor of the conventional nitrogenase.…”

Section: I]mentioning

confidence: 99%

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Comparative Biochemical Characterization of the Iron‐Only Nitrogenase and the Molybdenum Nitrogenase from Rhodobacter Capsulatus

Schneider

Gollan

Dröttboom

et al. 1997

European Journal of Biochemistry

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125

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The component proteins of the iron-only nitrogenase were isolated from Rhodobacter capsulatus (AnifhTDK, AmodABCD strain) and purified in a one-day procedure that included only one columnchromatography step (DEAE-Sephacel). This procedure yielded component 1 (FeFe protein, RclFe), which was more than 95% pure, and an approximately 80% pure component 2 (Fe protein, R c~~' ) . The highest specific activities, which were achieved at an R c~~V R C~~~ molar ratio of 40: 1, were 260 (C,H, from C,H,), 350 (NH, formation), and 2400 (H, evolution) nmol product formed . min-' . mg protein-'.The purified FeFe protein contained 26 -t 4 Fe atoms ; it did not contain Mo, V, or any other heterometal atom.The most significant catalytic property of the iron-only nitrogenase is its high H,-producing activity, which is much less inhibited by competitive substrates than the activity of the conventional molybdenum nitrogenase. Under optimal conditions for N, reduction, the activity ratios (mol N, reduced/mol H, produced) obtained were 1 : 1 (molybdenum nitrogenase) and 1 :7.5 (iron nitrogenase). The Rcl" protein has only a very low affinity for C,H,. The K,, value determined (12.5 kPa), was about ninefold higher than the K,, for RclM" (1.4 kPa). The proportion of ethane produced from acetylene (catalyzed by the iron nitrogenase), was strictly pH dependent. It corresponded to 5.5% of the amount of ethylene at pH 6.5 and was almost zero at pH values greater than 8.5.In complementation experiments, component 1 proteins coupled very poorly with the 'wrong' component 2. RclF', if complemented with R c~~" , showed only 10-15% of the maximally possible activity. Cross-reaction experiments with isolated polyclonal antibodies revealed that Rcl'" and Rc lM" are immunologically not related.The most active RclFe samples appeared to be EPR-silent in the Na,S,O,-reduced state. However, on partial oxidation with K,[Fe(CN),] or thionine several signals occurred. The most significant signal appears to be the one at g = 2.27 and 2.06 which deviates from all signals so far described for P clusters. It is a transient signal that appears and disappears reversibly in a redox potential region between -100 mV and + 150 mV. Another novel EPR signal (g = 1.96, 1.92, 1.77) occurred on further reduction of RclF" by using turnover conditions in the presence of a substrate (N,, C,H,, H+).Keywords: nitrogenase ; iron protein ; cofactor; EPR; Rhodobacter capsulatus.Three genetically distinct types of nitrogenase systems (ng vnJ unf, have so far been proved to exist in nature. The most widespread and intensively characterized system is the classical Mo-containing nitrogenase (nif system) found in all diazotrophs [l, 21. During the last few years, two types of alternative, Moindependent nitrogenases have been discovered and described. One is a vanadium-containing nitrogenase (vnf system) (for review see [3]) and the other enzyme lacks both Mo and V (anf system) and has been tentatively designated as Fe nitrogenase [4][5][6]. Although there is much circumstantial and...

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Molecular Metal Oxides in Protein Cages/Cavities

Müller

Rehder

2013

Coordination Chemistry in Protein Cages

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Detection of the in vivo incorporation of a metal cluster into a protein

Cited by 54 publications

References 34 publications

Reconstruction and minimal gene requirements for the alternative iron-only nitrogenase in Escherichia coli

Reconstruction and minimal gene requirements for the alternative iron-only nitrogenase in Escherichia coli

Comparative Biochemical Characterization of the Iron‐Only Nitrogenase and the Molybdenum Nitrogenase from Rhodobacter Capsulatus

Molecular Metal Oxides in Protein Cages/Cavities

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