Nitrogenase Chemistry at 10 Kelvin─Phototautomerization and Recombination of CO-Inhibited α-H195Q Enzyme.

CO-bound forms of nitrogenase are N2-reduction inhibited and likely intermediates in Fischer-Tropsch chemistry. Visible-light photolysis at 7 K was used to interrogate all three known CO-related EPR-active forms as exhibited by the α-H195Q variant of Azotobacter vinelandii nitrogenase MoFe protein. The hi(5)-CO EPR signal converted to the hi-CO EPR signal, which reverted at 10 K. FT-IR monitoring revealed an exquisitely light-sensitive "Hi-2" species with bands at 1932 and 1866 cm-1 that yielded "Hi-1" with bands at 1969 and 1692 cm-1, which reverted to "Hi-2". The similarities of photochemical behavior and recombination kinetics showed, for the first time, that hi-CO EPR and "Hi-1" IR signals arise from one chemical species. hi(5)-CO EPR and "Hi-2" IR signals are from a second species, and lo-CO EPR and "Lo-2" IR signals, formed after prolonged illumination, are from a third species. Comparing FT-IR data with CO-inhibited MoFe-protein crystal structures allowed assignment of CO-bonding geometries in these species.

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

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 have 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-cofactor (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 (Wang, S. Y. et al. Dalton Trans. 2018, 47, 7412-7421) 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 coexistence of homocitrates coordinated with α-alkoxy (deprotonated) and α-hydroxy (protonated) groups in the extracted FeMo-co.

Parallel inhibition of amino acid efflux and growth of erythrocytic Plasmodium falciparum by mefloquine and non-piperidine analogs: Implication for the mechanism of antimalarial action.

Despite the troubling psychiatric side-effects it causes in some patients, mefloquine (MQ) has been used for malaria prophylaxis and therapy, due to its activity against all Plasmodium species, its ease of dosing, and its relative safety in children and pregnant women. Yet at present there is no consensus on the mechanism of antimalarial action of MQ. Two leading hypotheses for the mechanism of MQ are inhibition of heme crystallization and inhibition of host cell hemoglobin endocytosis. In this report we show that MQ is a potent and rapid inhibitor of amino acid efflux from intact parasitized erythrocytes, which is a measure of the in vivo rate of host hemoglobin endocytosis and catabolism. To further explore the mechanism of action of MQ, we have compared the effects of MQ and 18 non-piperidine analogs on amino acid efflux and parasite growth. Among these closely related compounds, an excellent correlation over nearly 4 log units is seen for 50% inhibition concentration (IC50) values for parasite growth and leucine efflux. These data and other observations are consistent with the hypothesis that the antimalarial action of these compounds derives from inhibition of hemoglobin endocytosis.

Is Trehalose an Effective Quenching Agent of Azotobacter vinelandii Mo-Nitrogenase Turnover?

H2-evolution assays, plus EPR and FTIR spectroscopies, using CO-inhibited Azotobacter vinelandii Mo-nitrogenase have shown that the disaccharide trehalose is an effective quenching agent of enzymatic turnover and also stabilizes the reaction intermediates formed. Complete inhibition of H2-evolution activity was achieved at 1.5 M trehalose, which compares favorably to the requirement for 10 M ethylene glycol to achieve similar inhibition. Reaction-intermediate stabilization was demonstrated by monitoring the EPR spectrum of the 'hi-CO' form of CO-inhibited N2ase, which did not change during 1 hr after trehalose quenching. Similarly, in situ photolysis with FTIR monitoring of 'hi-CO' resulted in the same 1973 and 1681 cm-1 signals as observed previously in ethylene glycol-quenched systems. [a] These results clearly show that 1.5 M trehalose is an effective quench and stabilization agent for Mo-N2ase studies. Possible applications are discussed.

Structural characterization of CO-inhibited Mo-nitrogenase by combined application of nuclear resonance vibrational spectroscopy, extended X-ray absorption fine structure, and density functional theory: new insights into the effects of CO binding and the role of the interstitial atom.

The properties of CO-inhibited Azotobacter vinelandii (Av) Mo-nitrogenase (N2ase) have been examined by the combined application of nuclear resonance vibrational spectroscopy (NRVS), extended X-ray absorption fine structure (EXAFS), and density functional theory (DFT). Dramatic changes in the NRVS are seen under high-CO conditions, especially in a 188 cm(-1) mode associated with symmetric breathing of the central cage of the FeMo-cofactor. Similar changes are reproduced with the α-H195Q N2ase variant. In the frequency region above 450 cm(-1), additional features are seen that are assigned to Fe-CO bending and stretching modes (confirmed by (13)CO isotope shifts). The EXAFS for wild-type N2ase shows evidence for a significant cluster distortion under high-CO conditions, most dramatically in the splitting of the interaction between Mo and the shell of Fe atoms originally at 5.08 Å in the resting enzyme. A DFT model with both a terminal -CO and a partially reduced -CHO ligand bound to adjacent Fe sites is consistent with both earlier FT-IR experiments, and the present EXAFS and NRVS observations for the wild-type enzyme. Another DFT model with two terminal CO ligands on the adjacent Fe atoms yields Fe-CO bands consistent with the α-H195Q variant NRVS. The calculations also shed light on the vibrational "shake" modes of the interstitial atom inside the central cage, and their interaction with the Fe-CO modes. Implications for the CO and N2 reactivity of N2ase are discussed.

IR-monitored photolysis of CO-inhibited nitrogenase: a major EPR-silent species with coupled terminal CO ligands.

Fourier transform infrared spectroscopy (FTIR) was used to observe the photolysis and recombination of a new EPR-silent CO-inhibited form of α-H195Q nitrogenase from Azotobacter vinelandii. Photolysis at 4 K reveals a strong negative IR difference band at nu = 1938 cm(-1), along with a weaker negative feature at 1911 cm(-1). These bands and the associated chemical species have both been assigned the label "Hi-3". A positive band at nu = 1921 cm(-1) was assigned to the "Lo-3" photoproduct. By using an isotopic mixture of (12)C (16)O and (13)C (18)O, we show that the Hi-3 bands arise from coupling of two similar CO oscillators with one uncoupled frequency at approximately nu = 1917 cm(-1). Although in previous studies Lo-3 was not observed to recombine, by extending the observation range to 200-240 K, we found that recombination to Hi-3 does indeed occur, with an activation energy of approximately 6.5 kJ mol(-1). The frequencies of the Hi-3 bands suggest terminal CO ligation. This hypothesis was tested with DFT calculations on models with terminal CO ligands on Fe2 and Fe6 of the FeMo-cofactor. An S = 0 model with both CO ligands in exo positions predicts symmetric and asymmetric stretches at nu = 1938 and 1909 cm(-1), respectively, with relative band intensities of about 3.5:1, which is in good agreement with experiment. From the observed IR intensities, Hi-3 was found to be present at a concentration about equal to that of the EPR-active Hi-1 species. The relevance of Hi-3 to the nitrogenase catalytic mechanism and its recently discovered Fischer-Tropsch chemistry is discussed.

Carbon monoxide binding to α-R277H Mo-nitrogenase - Evidence for multiple pH-dependent species from IR-monitored photolysis.

The nitrogenase (N2ase) enzyme family is responsible for the conversion of dinitrogen into biologically accessible ammonia, a critical step in the global nitrogen cycle. Carbon monoxide (CO) has long been known as an inhibitor of dinitrogen reduction, but it can also be reduced to hydrocarbons catalyzed by all three N2ases, namely the wild-type Mo enzyme and select variants and the V and Fe nitrogenases, both of which are orders of magnitude more effective. CO interactions with N2ases are thus relevant to both dinitrogen fixation and Fischer-Tropsch-like chemistry. Here, we investigated the interaction of CO with the α-R277H variant of the Azotobacter vinelandii N2ase MoFe protein, in which the α-subunit 277Arg residue is replaced by His and results in production of only the S = 3/2 EPR signal (denoted as hi(5)-CO). Fourier-transform infrared (FT-IR) spectroscopy was used to follow the photolysis of CO bound to the α-R277H variant under cryogenic conditions. Multiple EPR-silent species were observed with FT-IR spectroscopic signatures previously assigned to CO-inhibited forms of the α-H195Q and α-H195N N2ase variants. The distribution of these CO-inhibited forms varied dramatically with pH over the range of pH 6.5 to pH 8.5, indicating protonation/deprotonation involvement.

Photolysis of Hi-CO Nitrogenase - Observation of a Plethora of Distinct CO Species using Infrared Spectroscopy.

Fourier transform infrared spectroscopy (FT-IR) was used to study the photochemistry of CO-inhibited Azotobacter vinelandii nitrogenase using visible light at cryogenic temperatures. The FT-IR difference spectrum of photolyzed hi-CO at 4 K comprises negative bands at 1973 cm-1 and 1679 cm-1 together with positive bands at 1711 cm-1, 2135 and 2123 cm-1. The negative bands are assigned to a hi-CO state that comprises 2 metal-bound CO ligands, one terminally bound, and one bridged and/or protonated species. The positive band at 1711 cm-1 is assigned to a lo-CO product with a single bridged and/or protonated metal-CO group. We term these species 'Hi-1' and 'Lo-1' respectively. The high-energy bands are assigned to a liberated CO trapped in the protein pocket. Warming results in CO recombination, and the temperature dependence of the recombination rate yields an activation energy of 4 kJ mol-1. Two α-H195 variant enzymes yielded additional signals. Asparagine substitution, α-H195N, gives a spectrum containing 2 negative 'Hi-2' bands at 1936 and 1858 cm-1 with a positive 'Lo-2' band at 1780 cm-1, while glutamine substitution, α-H195Q, produces a complex spectrum that includes a third CO species, with negative 'Hi-3' bands at 1938 and 1911 cm-1 and a positive feature 'Lo-3' band at 1921 cm-1. These species can be assigned to a combination of terminal, bridged, and possibly protonated CO groups bound to the FeMo-cofactor active site. The proposed structures are discussed in terms of both CO inhibition and the mechanism nitrogenase catalysis. Given the intractability of observing nitrogenase intermediates by crystallographic methods, IR-monitored photolysis appears to be a promising and information-rich probe of nitrogenase structure and chemistry.

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

Protonation of FeMo-cofactor is important for the process of substrate hydrogenation. Its structure has been clarified as Δ-Mo*Fe7S9C(R-homocit*)(cys)(Hhis) for the efforts of nearly 30 years, while it remains controversial whether FeMo-cofactor is protonated or deprotonated with chelated ≡C-O(H) homocitrate. We have used protonated molybdenum(V) lactates 1 and its enantiomer as model compounds for R-homocitrate in FeMo-cofactor of nitrogenase. Vibrational circular dichroism (VCD) spectrum of 1 at 1051 cm-1 is attributed to ≡C-OH vibration, and molybdenum(VI) R-lactate at 1086 cm-1 is assigned as ≡C-O α-alkoxy vibration. These vibrations set up labels for the protonation state of coordinated α-hydroxycarboxylates. The characteristic VCD band of NMF-extracted FeMo-cofactor is assigned to ν(C-OH), which is based on the comparison of molybdenum(VI) R-homocitrate. Density Functional Theory calculations are in consistent with these assignments. To the best of our knowledge, this is the first time that protonated R-homocitrate in FeMo-cofactor is confirmed by VCD spectra.

Low frequency dynamics of the nitrogenase MoFe protein via femtosecond pump probe spectroscopy - Observation of a candidate promoting vibration.

We have used femtosecond pump-probe spectroscopy (FPPS) to study the FeMo-cofactor within the nitrogenase (N2ase) MoFe protein from Azotobacter vinelandii. A sub-20-fs visible laser pulse was used to pump the sample to an excited electronic state, and a second sub-10-fs pulse was used to probe changes in transmission as a function of probe wavelength and delay time. The excited protein relaxes to the ground state with a ~1.2ps time constant. With the short laser pulse we coherently excited the vibrational modes associated with the FeMo-cofactor active site, which are then observed in the time domain. Superimposed on the relaxation dynamics, we distinguished a variety of oscillation frequencies with the strongest band peaks at ~84, 116, 189, and 226cm(-1). Comparison with data from nuclear resonance vibrational spectroscopy (NRVS) shows that the latter pair of signals comes predominantly from the FeMo-cofactor. The frequencies obtained from the FPPS experiment were interpreted with normal mode calculations using both an empirical force field (EFF) and density functional theory (DFT). The FPPS data were also compared with the first reported resonance Raman (RR) spectrum of the N2ase MoFe protein. This approach allows us to outline and assign vibrational modes having relevance to the catalytic activity of N2ase. In particular, the 226cm(-1) band is assigned as a potential 'promoting vibration' in the H-atom transfer (or proton-coupled electron transfer) processes that are an essential feature of N2ase catalysis. The results demonstrate that high-quality room-temperature solution data can be obtained on the MoFe protein by the FPPS technique and that these data provide added insight to the motions and possible operation of this protein and its catalytic prosthetic group.