Preliminary Assignment of Protonated and Deprotonated Homocitrates in Extracted FeMo-Cofactors by Comparisons with Molybdenum(IV) Lactates and Oxidovanadium Glycolates

Abstract

A similar pair of protonated and deprotonated mononuclear oxidovanadium glycolates [VO(Hglyc)(phen)(H2O)]Cl·2H2O (1) and [VO(glyc)(bpy)(H2O)] (2) and a mixed-(de)protonated oxidovanadium triglycolate (NH4)2[VO(Hglyc)2(glyc)]·H2O (3) were isolated and examined. The ≡C–O(H) (≡C–OH or ≡C–O) groups coordinated to vanadium were spectroscopically and structurally identified. The glycolate in 1 features a bidentate chelation through protonated α-hydroxy and α-carboxy groups, whereas the glycolate in 2 coordinates through deprotonated α-alkoxy and α-carboxy groups. The glycolates in 3 coordinate to vanadium through α-alkoxy or α-hydroxy and α-carboxy groups, and thus has both protonated ≡C–OH and deprotonated ≡C–O bonds simultaneously. Structural investigations revealed that the longer protonated V–Oα-hydroxy bonds [2.234(2) Å and 2.244(2) Å] in 1 and 3 are close to those of FeV-co 2.17 Å1 (FeMo-co 2.17 Å2), while deprotonated V–Oα-alkoxy bonds [2, 1.930(2); 3, 1.927(2) Å] were obviously shorter. This shows a similar elongated trend as the Mo–O distances in the previously reported deprotonated vs. protonated molybdenum lactates,3 and these vanadium and molybdenum complexes have the same local V/Mo-homocitrate structures as those of FeV/Mo-cos of nitrogenases. The IR spectra of these oxidovanadium and the previously synthesized molybdenum complexes including different substituted ≡C–O(H) model compounds show red-shifts for ≡C–OH vs. ≡C–O alternation, which further assign the two IR bands of extracted FeMo-co at 1084 and 1031 cm−1 to ≡C–O and ≡C–OH vibrations respectively. Although the structural data or IR spectra for some of the previously synthesized Mo/V complexes and extracted FeMo-co were measured earlier, this is the first time that the ≡C–O(H) coordinated peaks are assigned. The overall structural and IR results well suggest the co-existence of homocitrates coordinated with α-alkoxy (deprotonated) and α-hydroxy (protonated) groups in the extracted FeMo-co.

Assignment of protonated R-homocitrate in extracted FeMo-cofactor of nitrogenase via vibrational circular dichroism spectroscopy

“…25 for comparison. The C-O stretching frequencies of molybdenum R-homocitrate 21 can be served as references for C-OH vibrations in FeMo-co 20, and also those from the previous assignments of infrared spectra 20 . Here VCD spectra are much more sensitive only for the local environments around the chiral organic component of R-homocitrate and metal center of molybdenum atom than those from infrared spectra.…”

“…In our previous report, IR spectra of some alcohols, α-hydroxycarboxylic acids have been compared with Spectral Database for Organic Compounds (SDBS), where peak 1047 cm −1 is assigned to C-O H vibration for lactic acid in Supplementary Fig. 24 20 . Here strong VCD peaks at 1051 in 1 and 1053 cm −1 in 2 are assigned to the protonated C-OH stretching vibrations numbered as e in Fig.…”

“…To identify the protonation state of homocitrate in FeMo-co, we have measured the VCD spectrum of extracted FeMo-co Δ-Mo * Fe 7 S 9 C[R-(H)homocit * ](NMF) 2 (20) purified from nitrogenase in Azotobacter vinelandii, where coordination sites of histidine and cysteine residues have been substituted by Nmethylformamides. The VCD spectrum of FeMo-co is compared with that of the synthetic deprotonated molybdenum(VI) R- Fig.…”

“…With a computational study for a proposed protonated state of FeMo-co 18 , structural comparisons of oxidomolybdenum(IV) complexes with the local structures of FeMo-cofactors in Protein Data Bank (PDB) gave an indirect evidence for the protonation of α-alkoxy group in R-homocitrate 19 . Comparisons of infrared spectroscopy (IR) between FeMo-co and a series of model compounds provided a direct evidence for the two possibilities of αalkoxy and/or α-hydroxy groups in R-homocitrate 20 . Moreover, quantum mechanics/molecular mechanics calculations recommended protonation of α-alkoxy group in FeMo-co [21][22][23][24] .…”

“…Experimental and calculated VCD spectra of 20. Comparison of the experimental and calculated VCD spectra for NMF-extracted FeMo-co Δ-Mo * Fe 7 S 9 C[R-(H)homocit * ](NMF) 2(20).…”