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

Abstract

In vitro synthesis of the iron-molybdenum cofactor (FeMo-co) of dinitrogenase using homocitrate and its analog allows the formation of modified forms of FeMo-co that show altered substrate specificities (N2, acetylene, cyanide, or proton reduction) ofnitrogenase [reduced ferredoxin:dinitrogen oxidoreductase (ATP-hydrolyzing), EC 1.18.6.1]. The (LR,2S)-threo-and (1S,2S)-erythro-fluorinated diastereomers of homocitrate have been incorporated in vitro into dinitrogenase in place of homocitrate. Dinitrogenase activated with FeMo-co synthesized using threo-fluorohomocitrate reduces protons, cyanide, and acetylene but cannot reduce N2. In addition, proton reduction is inhibited by carbon monoxide (CO), a characteristic of dinitrogenase from NifV-mutants. Dinitrogenase activated with FeMo-co synthesized using erythro-fluorohomocitrate reduces protons, cyanide, acetylene, and N2. In this case proton reduction is not inhibited by CO, a characteristic of the wild-type enzyme. Cyanide reduction properties ofdinitrogenase activated with FeMo-co containing either fluorohomocitrate diastereomer are similar, and CO strongly inhibits cyanide reduction. Dinitrogenases activated with FeMo-co containing homocitrate analogs with a hydroxyl group on the C-1 position are much less susceptible to CO inhibition of cyanide reduction. However, proton and cyanide reduction by dinitrogenase containing FeMo-co activated with (1R,2S) threo-isocitrate is only one-third that of dinitrogenase activated with the racemic mixture of isocitrate and shows strong CO inhibition ofsubstrate reduction. These results suggest that CO inhibition of proton and cyanide reduction occurs when the hydroxyl group on the C-1 position of analogs is "trans" to the C-2 carboxyl group (i.e., in the threo conformation). When racemic mixtures ofthese analogs are used in the system, it seems that the erythro form is preferentially incorporated into dinitrogenase. Finally, carbonyl sulfide inhibition of substrate reduction by dinitrogenase is dependent on the homocitrate analog incorporated into FeMo-co.

FeMo cofactor maturation on NifEN

“…Upon activation by NifEN complete , MoFe protein shows not only C 2 H 4 -formation but also H 2 -formation and N 2 -fixation activities that are comparable with those resulting from the complete FeMoco maturation assay (Table 2). Although activities in these maturation assays are lower than those resulting from activation of ⌬nifB MoFe protein with isolated FeMoco (13), the activities for all substrates are proportionally reduced, which would not be expected if the cofactor were structurally perturbed or homocitrate was incor- rectly bound (25 (Table 2). In addition, the requirement of electron transfer for cluster conversion is suggested by the inability of NifEN apo Fe protein , prepared by replacing wild-type Fe protein with cluster-deficient apo Fe protein (Table 3), to reconstitute the ⌬nifB MoFe protein ( Table 2).…”

Purification of the Azotobacter vinelandii nifV-encoded homocitrate synthase

“…It might also be possible to use substituted forms or analogs of the homocitrate precursors to form homocitrate analogs. The availability of homocitrate analogs, as well as of radioactively labeled homocitrate, would be useful in expanding the approach described by Madden et al (11,12) to monitor the fate of homocitrate during FeMo cofactor formation and to determine the role of homocitrate in nitrogenase catalysis. (iii) Although it is clear that NifV catalyzes homocitrate formation, it is not yet known how Mo becomes coordinated to homocitrate, nor is it known how homocitrate and Mo become incorporated into FeMo cofactor.…”

Effects of homocitrate, homocitrate lactone, and fluorohomocitrate on nitrogenase in NifV- mutants of Azotobacter vinelandii

“…Homocitrate Madden et al (16) showed that dinitrogenase activated with FeMo-co synthesized in vitro by using erythrofluorohomocitrate effectively reduces protons (without CO inhibition), cyanide, acetylene, and N2 in a manner similar to that of homocitrate-activated dinitrogenase. However, erythro-fluorohomocitrate was unable to cure the NifVphenotype in vivo.…”

“…The solution was stored overnight at 5°C, and the pH was adjusted to 8 before use. erythro-Fluorohomocitrate was synthesized by the method of Madden et al (16). cFigures in parentheses are percent inhibition by CO. One hundred microliters of CO gas was injected into an assay vial containing an 8-ml gas phase.…”

“…to produce modified forms of FeMo-co (6,11,16). Dinitrogenases containing these aberrant forms of FeMo-co exhibit altered substrate specificity and inhibitor susceptibility.…”