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

Abstract

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...

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

“…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.…”

Overlapping and Specialized Functions of the Molybdenum-Dependent Regulators MopA and MopB in Rhodobacter capsulatus

“…Although expression of the morAB and morC genes is controlled by molybdenum, at present it remains unknown whether the gene products are involved in Mo uptake at all. The presence of the high-affinity Mo transporter ModABC, which provides sufficient Mo for the Mo-nitrogenase at low Mo concentrations, is physiologically favorable, as Mo-nitrogenase is more efficient than Fe-nitrogenase with respect to N 2 reduction rates (24).…”

Coordinated Expression of fdxD and Molybdenum Nitrogenase Genes Promotes Nitrogen Fixation by Rhodobacter capsulatus in the Presence of Oxygen

“…In addition to Mo-dependent nitrogenase, some diazotrophs synthesize alternative Mo-independent nitrogenases, the V-and Fe-nitrogenases, containing either an iron-vanadium or an iron-only cofactor, respectively (2). Mo-nitrogenase exhibits higher specific N 2 -reducing activity than Mo-free nitrogenases, and thus, Mo-nitrogenase is the preferred enzyme, as long as sufficient concentrations of molybdate are available (3,4). When molybdate, the only bioavailable form of molybdenum, becomes limiting in the environment, bacteria synthesize high-affinity ModABC transporters to import molybdate against the concentration gradient (5).…”