Characterization of an M-Cluster-Substituted Nitrogenase VFe Protein

Abstract

The Mo- and V-nitrogenases are two homologous members of the nitrogenase family that are distinguished mainly by the presence of different heterometals (Mo or V) at their respective cofactor sites (M- or V-cluster). However, the V-nitrogenase is ~600-fold more active than its Mo counterpart in reducing CO to hydrocarbons at ambient conditions. Here, we expressed an M-cluster-containing, hybrid V-nitrogenase in Azotobacter vinelandii and compared it to its native, V-cluster-containing counterpart in order to assess the impact of protein scaffold and cofactor species on the differential reactivities of Mo- and V-nitrogenases toward CO. Housed in the VFe protein component of V-nitrogenase, the M-cluster displayed electron paramagnetic resonance (EPR) features similar to those of the V-cluster and demonstrated an ~100-fold increase in hydrocarbon formation activity from CO reduction, suggesting a significant impact of protein environment on the overall CO-reducing activity of nitrogenase. On the other hand, the M-cluster was still ~6-fold less active than the V-cluster in the same protein scaffold, and it retained its inability to form detectable amounts of methane from CO reduction, illustrating a fine-tuning effect of the cofactor properties on this nitrogenase-catalyzed reaction. Together, these results provided important insights into the two major determinants for the enzymatic activity of CO reduction while establishing a useful framework for further elucidation of the essential catalytic elements for the CO reactivity of nitrogenase.IMPORTANCE This is the first report on the in vivo generation and in vitro characterization of an M-cluster-containing V-nitrogenase hybrid. The "normalization" of the protein scaffold to that of the V-nitrogenase permits a direct comparison between the cofactor species of the Mo- and V-nitrogenases (M- and V-clusters) in CO reduction, whereas the discrepancy between the protein scaffolds of the Mo- and V-nitrogenases (MoFe and VFe proteins) housing the same cofactor (M-cluster) allows for an effective assessment of the impact of the protein environment on the CO reactivity of nitrogenase. The results of this study provide a first look into the "weighted" contributions of protein environment and cofactor properties to the overall activity of CO reduction; more importantly, they establish a useful platform for further investigation of the structural elements attributing to the CO-reducing activity of nitrogenase.

Keywords: carbon monoxide; cofactor; hydrocarbons; molybdenum; nitrogenase; vanadium.

FIG 1

Subunit and metal compositions of VnfDGK^V and VnfDGK^M. (A) SDS-PAGE analysis of VnfDGK^V and VnfDGK^M. The molecular masses (in kilodaltons) of the protein standards are shown to the left of the gel. (B) Metal contents of NifDK^M, VnfDGK^V, and VnfDGK^M.

FIG 2

Spectroscopic and catalytic properties of the M-cluster extracted from VnfDGK^M. (A and B) EPR spectra (A) and activity profiles (B) of the cofactor-deficient NifDK^apo protein reconstituted with the M-cluster extracted from NifDK (NifDK^apo + M^NifDK) or VnfDGK^M (NifDK^apo + M^VnfDGK). The g values are indicated in panel A. Activities are expressed as nanomoles of product per nanomole of cofactor per min in panel B.

FIG 3

Spectroscopic and catalytic properties of NifDK^M, VnfDGK^V, and VnfDGK^M. (A and B) EPR spectra (A) and activity profiles (B) of NifDK^M, VnfDGK^V, and VnfDGK^M. Note the presence of the same S = 1/2 signal in the spectra of VnfDGK^V and VnfDGK^M (see panel A), which was assigned to the P*-cluster (i.e., a pair of [Fe₄S₄]-like clusters) in the case of VnfDGK^V (11). The g values are indicated in panel A, and the products are color coded in panel B. The substrates are indicated at the bottom of the bar chart, and the ratios of N₂/H₂ and C₂H₄/H₂ generated in the reactions of N₂ and C₂H₂ reduction are indictedabove the respective bars in panel B. HCs, hydrocarbons.

FIG 4

CO-reducing activities and product profiles of NifDK^M, VnfDGK^V, and VnfDGK^M. (A to C) Individual activities of hydrocarbon formation (A), distributions of hydrocarbon products (B), and total activities of hydrocarbon product formation (C) by NifDK^M, VnfDGK^V, and VnfDGK^M when CO is supplied as a substrate. Activities are expressed as nanomoles of product per nanomole of cofactor per minute in panels A and C. The percentage activities of proteins are shown in red in panel C, with the total activity of VnfDGK^V set at 100% and those of NifDK^M and VnfDGK^M calculated accordingly. Note that there is a contribution of V to the activity of VnfDGK^M due to the presence of <0.07 nmol of V per nmol of VnfDGK^M. However, the percentage contribution of V to the overall activity of VnfDGK^M cannot be conclusively determined due to the inaccuracy of V determination in this low concentration range.