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

Abstract

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.

Figure 1.

Overlays of FeMo-cofactor environments in wild-type (WT) (PDB 3U7Q) [5] and α-H195Q (PDB 1FP4) [6] MoFe proteins with the locations of one CO (PDB 4TKV) [7] or two CO (PDB 7JRF) [8] ligands. With similar bridging CO positioning, for clarity, only the last structure is shown.

Figure 2.

Time-dependent EPR- and IR-monitored photolysis of CO-inhibited α-H195Q N2ase. EPR g-values are color-coded: hi(5)-CO (red), hi-CO (blue), lo-CO (black), resting N2ase (green). Spectra are also color-coded according to their position in time sequence. A. EPR in g=5 region with 700- or 550-nm light. Times for 700 nm: 0 s, 5 min, 20 min, 40 min; times for 550 nm: 0 s, 40 s, 4 min, 130 min. B. Changes in EPR in g=2 region with 450-nm or 700-nm light. Times for 450 nm: 5 min (black), 15 min (red), 35 min (green), 60 min (blue); times for 700 nm: 5 min (black), 10 min (red), 30 min (green), 60 min (blue). C. IR difference spectra with 410-nm or 700-nm light. Times for 410 nm: 2 s (black), 7 s (red), 11 s (blue), 30 s (green), 15 min (black), 25 min (red), 30 min (blue), 40 min (green). Times for 700 nm: 20 s (black), 60 s (red), 3 min (blue), 40 min (green). IR frequencies are color-coded: ‘Hi-1’ (blue), ‘Hi-2’ (red), ‘Lo-2’ (black).

Figure 3.

A. Stopped-flow FT-IR spectra for 1:1 mixing of α-H195Q MoFe protein and Fe protein with a buffered solution of MgATP saturated with 1 atm CO. B. Recombination kinetics for the hi(5)-CO EPR signal as a function of temperature for samples in H2O (x) or D2O (o) buffer. Smooth curves correspond to stretched exponential fits. C. Recombination of the ‘Hi-1’→’Hi-2’ IR signals at 12K. The ΔA refers to a pre-photolysis baseline. Times illustrated are: 10, 19, 29, 38, 96, 191, and 239 s.