Purification and characterization of a FeMo cofactor-deficient MoFe protein

Abstract

Previous studies have shown that the nifH gene product is required for FeMo cofactor biosynthesis and insertion and that a delta nifH strain of Azotobacter vinelandii designated DJ54 accumulates a FeMo cofactor-deficient MoFe protein that is distinct from the FeMo cofactor-deficient protein synthesis by Nif B-, N-, or E- strains [Tal, S., Chun, T., Gavini, N., & Burgess, B. K. (1991) J. Biol. Chem. 266, 10654-10657]. Here we report the purification and activation of the MoFe protein from DJ54. The purified protein is an alpha 2 beta 2 tetramer that is indistinguishable from the wild-type MoFe protein by the criteria of SDS-polyacrylamide gel electrophoresis, native gel electrophoresis, and two-dimensional gel electrophoresis. It binds normally to its redox partner, the Fe protein, by the criterion of chemical cross-linking. It does not contain FeMo cofactor and does not catalyze significant C2H2 reduction or reduction-independent MgATP hydrolysis. It can, however, be activated with FeMo cofactor following the addition of the Fe protein and MgATP when an additional required component(s) is supplied by cell-free extracts from a delta nifD strain of A. vinelandii. The purified DJ54 MoFe protein does contain P-clusters by the criteria of metal analysis, CD spectroscopy, cluster extrusion, and electrochemical reduction of the POX state. In the presence of dithionite it exhibits an axial S = 1/2 EPR signal that integrates to 0.1-0.3 spin per alpha 2 beta 2 tetramer.(ABSTRACT TRUNCATED AT 250 WORDS)

Construction of a Form of the MoFe Protein of Nitrogenase That Accepts Electrons from the Fe Protein but Does Not Reduce Substrate

“…Second, as shown in Fig. 3, the intensity of the visible spectrum is now very similar to that obtained for the wild-type MoFe protein whereas the FeMo cofactor-deficient MoFe protein purified from DJ54 has greatly reduced absorbance (18). Third, the circular dichroism spectra of the wild-type protein and the MoFe cluster-containing protein show two features at 530 and 660 nm (Fig.…”

“…For DJ54, the MoFe protein peak is greatly reduced in size because the protein has only 54% of the Fe of the wild-type protein due to the absence of FeMo cofactor centers (Ref. 18 and Fig. 1).…”

“…Materials-The wild-type Azotobacter vinelandii Fe and MoFe proteins (20), the MoFe cluster (19), and the FeMo cofactor-deficient MoFe protein from ⌬nifH strain DJ54 (18) were purified and analyzed as described elsewhere. DEAE-cellulose 52 was from Whatman, Q-Sepharose was from Pharmacia, and AcA34 was from Bio Sepra.…”

“…For protein purification cells were grown as described previously (18). Large scale insertion of MoFe cluster into the FeMo cofactor-deficient MoFe protein and subsequent purification of the protein product are described in the text.…”

“…We wanted to construct a form of the MoFe protein that could accept electrons from the Fe proteinMgATP complex but that could not reduce H ϩ . We have previously reported the purification and characterization of a FeMo cofactor-deficient form of the MoFe protein that contains Pclusters (18). In a separate study we also reported the large scale isolation and characterization of a species designated the MoFe cluster that contains the metal portion of FeMo cofactor that gives rise to the S ϭ 3/2 EPR signal but which appears to be missing homocitrate and does not support any substrate reduction (19).…”