Purification, Characterization And Crystal Growth Of MoFe Protein And HBP59 From A NifE-deletion Strain DJ35 Of Azotobacter Vinelandii

A FeMo cofactor-deficient MoFe protein was purified from a △nifE strain of Azotobacter vinelandii designated DJ35. △nifE MoFe protein exhibited two kinds of behavior of native electrophoresis. It contained 0.36 molecule Mo and 9.96 molecule Fe per molecule of △nifE Av1. The absorption spectra were similar for △nifE Av1 and OP Av1, showing a broad decrease in absorbance in the 300 nm-600 nm region and no obvious features. CD spectra indicated that a major feature present at ca.450 nm was similar quantitatively and qualitatively when △nifE Av1 was compared with OP Av1. In contrast, the feature at 500 nm-700 nm was much smaller in △nifE Av1. In comparison with the reduced OP Av1, △nifE Av1 completely lost the EPR signal at g≈3.7, while the intensity at g≈4.3 and 2.0 were significantly reduced. △nifE Av1 had no C2H2 reduction activity when complemented with Fe protein, but could be activated by FeMoco extracted from Av1. The above results indicated that △nifE Av1 may be a FeMoco-deficient protein containing P-cluster. Besides these, the optimum conditions of crystal growth for △nifE Av1 and △nifH Av1 were selected and single crystals of △nifE Av1 and △nifH Av1 were obtained. Attempting to obtained △nifE Av1,we found a contaminant protein similar to subunit of OP Av1 in relative mobility. The analysis of MALDI TOF MS showed that it was the product of a predicted gene in A.vinelandii. Modifying the purification procedure, we had, at first, obtained the new protein with 80% purity and the new protein was termed HBP59. Initial characterizations of were investigated. The protein was monomer with apparent molecular weight of ≈ 59 kDa. The metal analysis showed that HBP59 contained 0.42 molecule Fe per molecule of protein. UV/visible absorption spectra of HBP59 were similar to that of major heme-binding proteins, showing three peaks at 421 nm, 517 nm and 556 nm in Soret region in the reduced state, respectively. The maximal peak was shifted from 421 nm to 413 nm after exposure to air. Soret CD positive peaks of HBP59 in reduced state were presented at 420 nm, 406 nm, 379 nm and 364 nm with intensity 0.75 M-1 cm-1, 0.94 M-1 cm-1, 0.68 M-1 cm-1 and 0.99 M-1 cm-1,respectively, while negative peaks at 433 nm and 392 nm with intensity -1.13 M-1 cm-1 and -0.78 M-1 cm-1. The titration of heme showed that the protein had 0.1 heme molecule per molecule of protein, but could interact with heme in a 1:1 molar ratio. The low ratio of A421 nm to A280 nm (0.146) was consistent with the low content of heme. The above results indicated that HBP59 had the asymmetric heme with low spin and maybe involved in heme utilization or adhesion as predicted. Besides these, the optimum conditions of crystal growth for HBP59 were selected and single crystals of HBP59 were obtained.