Rapid Purification of the Protein Components of a Highly Active ?Iron Only? Nitrogenase
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In the presence of molybdate (1 μM) 2–3.5% oxygen and with sucrose as carbon source, Xanthobacter autotrophicus GZ29, a microaerophilic nitrogen‐fixing hydrogen‐oxidizing bacterium, grew diazotrophically with a minimal doubling time of 2.5 h and a calculated absorbance of up to 52 (546 nm). The maximal specific activity obtained was 145 nmol ethylene reduced ċ min−1· mg protein−1 (crude extract). The Mo nitrogenase was derepressed to a comparable level with methionine as nitrogen source. Vanadium compounds stimulated neither growth nor nitrogenase activity. Without added molybdate, diazotrophic growth and nitrogenase activity decreased to an extremely low level. The nitrogenase, responsible for the residual activity in molybdate‐starved cells, contained molybdate but no other heterometal atom. These results indicate that, in X. autotrophicus, a Mo‐independent nitrogenase does not exist. However, the molybdate‐containing nitrogenase exhibited some properties which are reminiscent of alternative nitrogenases. The MoFe protein (component 1, Xa1) copurified with two molecules of a small, not previously detected polypeptide (molar mass 13.6 kDa) and was able to reduce acetylene not only to ethylene but also partly to ethane. Under certain conditions, i.e. in Tris/HCl buffer at alkaline pH values, with titanium (III) citrate as electron donor, at high component 1/component 2 ratios, and at low, non‐saturating acetylene concentrations, up to 5.5% ethane was measured. Parallel to the pH‐dependent increase of the relative yield of ethane, the total activity (both acetylene and nitrogen reduction rates) decreased and the S =3/2 FeMo cofactor ESR signal was split into three signals with different rhombicities [E/D values of 0.036 (signal I), 0.072 (signal II) and 0.11 (signal III)]. The intensities of the two new FeMo cofactor signals were more pronounced the more alkaline the pH. They could be further enhanced using titanium (III) citrate instead of Na2S2O4 as reductant.
In the presence of molybdate (1 μM) 2–3.5% oxygen and with sucrose as carbon source, Xanthobacter autotrophicus GZ29, a microaerophilic nitrogen‐fixing hydrogen‐oxidizing bacterium, grew diazotrophically with a minimal doubling time of 2.5 h and a calculated absorbance of up to 52 (546 nm). The maximal specific activity obtained was 145 nmol ethylene reduced ċ min−1· mg protein−1 (crude extract). The Mo nitrogenase was derepressed to a comparable level with methionine as nitrogen source. Vanadium compounds stimulated neither growth nor nitrogenase activity. Without added molybdate, diazotrophic growth and nitrogenase activity decreased to an extremely low level. The nitrogenase, responsible for the residual activity in molybdate‐starved cells, contained molybdate but no other heterometal atom. These results indicate that, in X. autotrophicus, a Mo‐independent nitrogenase does not exist. However, the molybdate‐containing nitrogenase exhibited some properties which are reminiscent of alternative nitrogenases. The MoFe protein (component 1, Xa1) copurified with two molecules of a small, not previously detected polypeptide (molar mass 13.6 kDa) and was able to reduce acetylene not only to ethylene but also partly to ethane. Under certain conditions, i.e. in Tris/HCl buffer at alkaline pH values, with titanium (III) citrate as electron donor, at high component 1/component 2 ratios, and at low, non‐saturating acetylene concentrations, up to 5.5% ethane was measured. Parallel to the pH‐dependent increase of the relative yield of ethane, the total activity (both acetylene and nitrogen reduction rates) decreased and the S =3/2 FeMo cofactor ESR signal was split into three signals with different rhombicities [E/D values of 0.036 (signal I), 0.072 (signal II) and 0.11 (signal III)]. The intensities of the two new FeMo cofactor signals were more pronounced the more alkaline the pH. They could be further enhanced using titanium (III) citrate instead of Na2S2O4 as reductant.
The Molybdenum Nitrogenase from Wild-type Xanthobacter autotrophicus Exhibits Properties Reminiscent of Alternative Nitrogenases
In the presence of molybdate (1 pM) 2-3.5% oxygen and with sucrose as carbon source, Xanthobacter autotrophicus GZ29, a microaerophilic nitrogen-fixing hydrogen-oxidizing bacterium, grew diazotrophically with a minimal doubling time of 2.5 h and a calculated absorbance of up to 52 (546 nm).The maximal specific activity obtained was 145 nmol ethylene reduced . min-' . mg protein-' (crude extract). The Mo nitrogenase was derepressed to a comparable level with methionine as nitrogen source. Vanadium compounds stimulated neither growth nor nitrogenase activity. Without added molybdate, diazotrophic growth and nitrogenase activity decreased to an extremely low level. The nitrogenase, responsible for the residual activity in molybdate-starved cells, contained molybdate but no other heterometal atom. These results indicate that, in X . autotrophicus, a Mo-independent nitrogenase does not exist. However, the molybdate-containing nitrogenase exhibited some properties which are reminiscent of alternative nitrogenases.The MoFe protein (component 1, Xal) copurified with two molecules of a small, not previously detected polypeptide (molar mass 13.6 kDa) and was able to reduce acetylene not only to ethylene but also partly to ethane. Under certain conditions, i.e. in Tris/HCl buffer at alkaline pH values, with titanium (111) citrate as electron donor, at high component l/component 2 ratios, and at low, non-saturating acetylene concentrations, up to 5.5% ethane was measured. Parallel to the pH-dependent increase of the relative yield of ethane, the total activity (both acetylene and nitrogen reduction rates) decreased and the S = 3/2 FeMo cofactor ESR signal was split into three signals with different rhombicities [ED values of 0.036 (signal I), 0.072 (signal 11) and 0.11 (signal III)]. The intensities of the two new FeMo cofactor signals were more pronounced the more alkaline the pH. They could be further enhanced using titanium (111) citrate instead of Na,S,O, as reductant.Keywords. Nitrogenase ; MoFe protein ; FeMo cofactor ; ethane formation; ESR.The 'classical' Mo-containing nitrogenase is a well-characterized N,-reducing enzyme system widespread in microorganisms [1]. It consists of two metalloproteins, component 1 (MoFe protein) and component 2 (Fe protein). The tetrameric MoFe protein (a2P2), on which we focus our main interest in this work, contains two types of unique metal clusters, the so-called 'P' cluster (Fe,S,-,) and the FeMo cofactor (FeMoco, 'M' cluster) which is considered to be the site of substrate binding and reduction. In recent publications by Rees and coworkers [2-41, structural models for both cluster types were proposed, based on crystallographic analyses of the MoFe protein from Azotobacter vinelandii, refined up to 0.22-nm resolution. FeMoco is located in the a subunit and consists of an Fe,S, and a MoFe,S, cluster fragment bridged by probably three p,-sulCorrespondence to A. Muller,
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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