Substrate reduction properties of dinitrogenase activated in vitro are dependent upon the presence of homocitrate or its analogs during iron-molybdenum cofactor synthesis

Abstract

(R)-2-Hydroxy-1,2,4-butanetricarboxylic acid [(R)-homocitrate] has been has been recently reported to be an integral constituent of the otherwise thought to be inorganic iron-molybdenum cofactor of dinitrogenase [Hoover, T.R., Imperial, J., Ludden, P.W., & Shah, V.K. (1989) Biochemistry 28,2768-2771]. Different organic acids can substitute for homocitrate in an in vitro system for iron-molybdenum cofactor synthesis and incorporation into dinitrogenase [Hoover, T.R., Imperial, J., Ludden, P.W., & Shah, V. K. (1988) Biochemistry 27, 3647-3652]. Dinitrogenase activated with homocitrate-FeMo-co was able to reduce dinitrogen, acetylene, and protons efficiently. Homoisocitrate and isocitrate dinitrogenases did not reduce dinitrogen or acetylene, but showed very high proton reduction activities. Citrate and citramalate dinitrogenases had very low dinitrogen reduction activities and intermediate acetylene and proton reduction activities. CO inhibited proton reduction in both these cases but not in the case of dinitrogenases activated with other homocitrate analogues. By use of these and other commercially available homocitrate analogues in the in vitro system, the structural features of the homocitrate molecule absolutely required for the synthesis of a catalytically competent iron-molybdenum cofactor were determined to be the hydroxyl group, the 1- and 2-carboxyl groups, and the R configuration of the chiral center. The stringency of the structural requirements was dependent on the nitrogenase substrate used for the assay, with dinitrogen having the most stringent requirements followed by acetylene and protons.

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

“…The component 1 protein of nitrogenases contains two types of unique metal clusters, the cofactor (FeMo cofactor, FeV cofactor, FeFe cofactor), also called M-cluster, which most likely represents the site of substrate reduction [16], and the P-cluster whose function is not yet definitely known, but whose primary [3, 5, 14, 151. role probably is to transfer electrons from the Fe protein to the cofactor [17]. In recent publications by Rees and co-workers and Bolin et al [20], structural models for both the FeMo cofactor (Fe,MoS&omocitrate) and the P-cluster (Fe8S,.J were proposed based on X-ray crystal structure analysis of the conventional nitrogenase MoFe protein from A. vinelundii and Clostridium pasteuriunum at up to 0.22 nm resolution.…”

Diastereomer-dependent substrate reduction properties of a dinitrogenase containing 1-fluorohomocitrate in the iron-molybdenum cofactor.

“…1) were tested for their ability to replace homocitrate in the FeMo-co synthesis system (11,13). The resulting variant forms of FeMo-co exhibited altered substrate specificity and inhibitor susceptibility.…”

Requirement of Homocitrate for the Transfer of a49V-Labeled Precursor of the Iron-Vanadium Cofactor from VnfX to nif-apodinitrogenase

“…The reactions were carried out under argon. The standard reaction mixture contained the following ingredients (first phase) in a total volume of 0.75 ml: 0.1 ml of 25 mM Tris-HCl (pH 7.4) containing 0.5 mM sodium dithionite, 20 l of 5 mM homocitrate (pH 8), 10 l of 1 mM Na 3 VO 4 , 0.2 ml of an ATP-regenerating mixture (27) containing 0.1 mM dithionite, NifB-co (0.75 nmol of iron), dinitrogenase reductase (52 g of protein), 0.2 ml each of the extracts to be tested (between 1.7 and 2.5 mg of protein), and, when stated, purified nifapodinitrogenase (34 g of protein). When reconstituted with an excess of purified FeMo-co, 34 g of purified nif-apodinitrogenase produced 36 nmol of ethylene/min, and the nif-apodinitrogenase present in 200 l of UW45 (W) extract produced 35 nmol of ethylene/min.…”

“…To test whether homocitrate incorporation into the unfinished cluster occurs in vitro, we studied the effect of homocitrate analogues on the reaction. These organic acids are able to replace homocitrate in the FeMo-co synthesis system (27,32), resulting in the synthesis of aberrant forms of FeMo-co that are known to exhibit altered substrate specificity (27,32). The use of isocitrate and malate in FeMo-co synthesis results in dinitrogenases with 8 -16% of the wild type acetylene reduction activities (27).…”

“…These organic acids are able to replace homocitrate in the FeMo-co synthesis system (27,32), resulting in the synthesis of aberrant forms of FeMo-co that are known to exhibit altered substrate specificity (27,32). The use of isocitrate and malate in FeMo-co synthesis results in dinitrogenases with 8 -16% of the wild type acetylene reduction activities (27). When added in an excess in in vitro FeMo-co synthesis assays containing homocitrate, these organic acids cause a reduction in the level of the acetylene reduction activity of the resulting dinitrogenase.…”