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

Abstract

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.

Keywords: Carbon monoxide; FT-IR; Nitrogen fixation; Nitrogenase; pH dependence; α-R277H-MoFe protein.

Figure 1.

Superimposed crystal structures with (A) a single bridging CO (PDB 4TKV) [6] or (B) terminal plus bridging CO (PDB 7JRF) [7]. Superimposed crystal structures with (C) wild-type enzyme (PDB 3U7Q) [11] vs. (D) approximate structure for the α-R277H variant.

Figure 2.

A. IR-difference spectra during broadband photolysis of ¹²CO- or ¹³CO-treated α-R277H at pH 7.4. Illustrated ¹²CO photolysis times were 0, 1, 5, 15, 45, and 120 min. Illustrated ¹³CO photolysis times were 0, 1, 5, 30, and 60 min. Arrows indicated photolysis time evolution of intensity. B. Photolysis results using 440 nm irradiation. Illustrated photolysis times were 0, 10, 20, 30, 45, 60, 90 120, and 180 min. C. A fit (−) to the final 440 nm photolysis data () with 5 Gaussians, central energies indicated in corresponding colors.

Figure 3.

A. Trapped photolyzed CO features for α-R277H compared with wild-type and α-H195Q N2ase. Dashed traces are before photolysis. B. Illustrative recombination curves for 60 K () and 100 K () intensity at 1937 cm⁻¹ and fits (−). Inset: corresponding Arrhenius plot including 40 K, 60 K, 80 K, 100 K, and 120 K.

Figure 4.

A. Comparison of IR-difference spectra during broadband photolysis of CO-bound α-R277H at pH 6.5, pH 7.4 and pH 8.5 (Δ Absorbance axis expanded 4-fold). Illustrated pH 6.5 and 7.4 photolysis times: 0, 5, 30, 60, 90, and 120 min; pH 8.5: 0, 5, 30, 60 and 90 min. B. Enlargements of smaller features, for the same traces.

Figure 5.

Minimal structures previously proposed for species detected in IR-monitored photolysis. *an unknown interaction lowers the CO stretching frequency; ‡ minor band; † coupled oscillators.

Figure 6.

A. One explanation for the multiple ‘Hi-2’ and ‘Lo-2’ bands observed for α-R277H CO signals at pH 7.4. B. One explanation for the multiple ‘Hi-2’ and ‘Lo-2’ bands observed for α-R277H CO signals at pH 8.5.