EXAFS and NRVS reveal a conformational distortion of the FeMo-cofactor in the MoFe nitrogenase propargyl alcohol complex

Abstract

We have used EXAFS and NRVS spectroscopies to examine the structural changes in the FeMo-cofactor active site of the α-70(Ala) variant of Azotobacter vinelandii nitrogenase on binding and reduction of propargyl alcohol (PA). The Mo K-edge near-edge and EXAFS spectra are very similar in the presence and absence of PA, suggesting PA does not bind at Mo. By contrast, Fe EXAFS spectra show a clear and reproducible change in the long Fe-Fe interaction at ~3.7 Å on PA binding with the apparent appearance of a new Fe-Fe interaction at 3.99 Å. An analogous change in the long Mo-Fe 5.1 Å interaction is not seen. The NRVS spectra exclude the possibility of large-scale structural change of the FeMo-cofactor involving breaking the μ(2) Fe-S-Fe bonds of the Fe(6)S(9)X core. The simplest chemically consistent structural change is that the bound form of PA is coordinated at Fe atoms (Fe6 or Fe7) adjacent to the Mo terminus, with a concomitant movement of the Fe away from the central atom X and along the Fe-X bond by about 0.35 Å. This study comprises the first experimental evidence of the conformational changes of the FeMo-cofactor active site on binding a substrate or product.

Figure 1

Mo K-edge x-ray absorption spectroscopy of α-70^Ala:PA compared with α-70^Ala:turnover and α-70^Ala:resting. (a) near-edge spectra, (b) near-edge 2^nd derivative spectra, (c) EXAFS spectra and (d) EXAFS Fourier transform spectra phase corrected for Mo-S. In each panel: upper spectrum - α-70^Ala:resting; center spectrum - α-70^Ala:turnover; lower spectrum - α-70^Ala:PA. In (c) and (d) broken line - data; solid line – fit.

Figure 2

Fe K-edge x-ray absorption spectroscopy of α-70^Ala:PA compared with α-70^Ala:turnover and α-70^Ala:resting. (a) near-edge spectra, (b) near-edge 2^nd derivative spectra, (c) EXAFS spectra and (d) EXAFS Fourier transform spectra phase corrected for Fe-S. In each panel: upper spectrum - α-70^Ala:resting; center spectrum - α-70^Ala:turnover; lower spectrum - α-70^Ala:PA. In (c) and (d) broken line - data; solid line - fit. (i) and (ii) in (d) are different fits for α-70^Ala:PA as described in the text.

Figure 3

NRVS results. (a) NRVS spectrum of α-70^Ala:PA (solid line) compared with resting wild-type AvI (broken line). (b) Calculated NRVS spectra showing the possible effect of opening the Fe₆S₉X core simulated by deleting S2b (solid line) compared with that calculated for unmodified FeMo-cofactor (broken line). The calculations do not include the P-cluster. The shaded region is discussed in the text.

Scheme 1

The structure of the FeMo-cofactor drawn from (PDB file 1M1N [3]). Mo – purple; Fe – brown; S – yellow, O – red; N – blue; C – grey; X – green. The Fe atoms are numbered in red.

Scheme 2

The FeMo-cofactor showing (a) structural movements considered in the text, (b) distances important for Mo EXAFS and (c) distances important for Fe EXAFS. The interstitial atom “X” is omitted for clarity in (b) and (c). The atoms colors are given in scheme 1.

Scheme 3

The FeMo-cofactor showing the proposed structure of the PA bound form consistent with the EXAFS and NRVS analysis. The atoms colors are given in scheme 1.