Turnover-Dependent Inactivation of the Nitrogenase MoFe-Protein at High pH

Abstract

Proton uptake accompanies the reduction of all known substrates by nitrogenase. As one consequence, higher pH should limit the availability of protons as a substrate essential for turnover, thereby increasing the proportion of more highly reduced forms of the enzyme for further study. The utility of the high pH approach would appear problematic in view of the observation reported by Pham and Burgess (Biochemistry 32, 13725 (1993)) that the MoFe-protein undergoes irreversible protein denaturation above pH 8.65. In contrast, we found by both enzyme activity and crystallographic analyses that MoFe-protein is stable when incubated at pH 9.5. We did observe, however, that at higher pHs and under turnover conditions, the MoFe-protein is slowly inactivated. While normal, albeit low, substrate reduction occurs under these conditions, the MoFe-protein undergoes a complex transformation; initially the enzyme is reversibly inhibited for substrate reduction at pH 9.5 yet in a second, slower process the MoFe-protein becomes irreversibly inactivated as measured by substrate reduction activity at the optimal pH of 7.8. The final inactivated MoFe-protein has an increased hydrodynamic radius compared to native MoFe-protein yet it has a full complement of iron and molybdenum. Significantly, the modified MoFe-protein retains the ability to specifically interact with its nitrogenase partner, the Fe-protein, as judged by the support of ATP hydrolysis and by tight complex formation with Fe-protein in the presence of ATP and aluminum fluoride. The turnover-dependent inactivation coupled to conformational change suggests a mechanism based transformation that may provide a new probe of the nitrogenase catalysis.

QM/MM Study of the Nitrogenase MoFe Protein Resting State: Broken-Symmetry States, Protonation States, and QM Region Convergence in the FeMoco Active Site

“…In previous studies 28,29,30,31,32 by one of us (RB) we have assigned the alkoxide group of the homocitrate as protonated but have not until now given a complete justification for this as the question of how well the protein environment is described surrounding the homocitrate remained unclear. The only exception to this is the 4ND8 crystal structure, recently published by Rees & Howard et al 85 which is a crystal structure solved under basic (pH 9.5) conditions, where the O-O distance is on average 2.77 Å as shown in Figure 6d. This is in very good agreement with a QM/MM model where the proton is missing (Figure 6d).…”

Influences of pH and organic carbon on oxalate removal by alkaliphilic biofilms acclimatized to nitrogen-deficient and supplemented conditions

“…This experiment hints at a strategy that could be effectively used to increase the biomass activity of an N-deficient biofilm oxidizing oxalate exposed to an unfavourable high pH such as pH 9. As previously highlighted, nitrogenase activity is compromised at high pH (Yang et al, 2014), and this likely imposes severe nitrogen limitation, affecting microbial growth. The exposure to favourable pH conditions increases nitrogenase activity reducing limitations of nitrogen for growth and as a consequence, a rapid increase of biomass can be anticipated.…”

“…In the absence of an inorganic source of N, the oxalate oxidizing activity recorded in the N-deficient reactor suggested that the microorganisms there would have to fulfil their N requirements plausibly via biological fixation of atmospheric N. It is well known that the enzyme responsible for catalysing the fixation of N is nitrogenase (Hernandez et al, 2009). This enzyme is known to be inhibited when exposed to highly alkaline conditions (Yang et al, 2014). According to Hadfield and Bulen (1969) and Igarashi et al (2005), the pH dependence of nitrogenase activity exhibits a bell-shaped relationship, whereby an optimal activity is at approximately pH 7-8.…”

“…According to Hadfield and Bulen (1969) and Igarashi et al (2005), the pH dependence of nitrogenase activity exhibits a bell-shaped relationship, whereby an optimal activity is at approximately pH 7-8. Therefore, an increase of pH beyond 8 significantly reduces activity, not due to high pH inactivation of MoFe-proteins in nitrogenase, but rather as a consequence of a complex, mechanism-based reaction (Yang et al, 2014).…”

Enantioselective synthesis of tungsten trimetallic cluster chalcogenides

“…In particular, molybdenum and tungsten heterometallic M 3 M'S 4 (M'= transition metal) sulfides have been proposed as models for metalloenzimes [1] or industrial hidrodesulfurization (M'= Ni or Co) catalysts [2,3]. Hidai et al…”