The Nitrogen‐Fixing System of Corynebacterium autotrophicum

Both nitrogenase components were isolated and purified from sucrose‐grown, nitrogen‐fixing cells of Corynebacterium autotrophicum by DEAE‐cellulose and Sephadex gel chromatography. The MoFe protein (Ca1) and the Fe protein (Ca2) appeared as single bands in sodium dodecylsulfate gel electrophoresis (Serva). Ca1 had an average molecular weight of 232000 with two non‐identical subunits ( M r 61200 and 57500) on Koch‐Light sodium dodecylsulfate gels; the Ca2 protein had an average molecular weight of 72600 with two identical subunits. Ca1 contained 2.2 mol Mo/mol, 23.1 mol Fe/mol and 20.1 mol S 2− /mol, the Ca2 protein 3.8 mol Fe/mol and 2.4 mol S 2− /mol. The amino acid composition of both nitrogenase proteins was similar to those of other N 2 ‐fixing bacteria except for the occurrence of an unidentified derivative. Electron paramagnetic resonance (EPR) spectroscopy of Ca1 protein gave a rhombic signal with g values of 4.13, 3.72 and 2.03 at pH 6.2 with small features to low field of the g = 4.13 peak and to high field of the g = 3.72 peak. At pH 9.25 the intensity of the EPR signal was transferred to minor features with g ‐values of 4.31 and 3.54 and of 4.68 and 3.31. Upon addition of acetylene to Ca1 at pH 9.25 the signal returned to the low‐pH form. The additional features were interpreted as being exhibited by two additional conformations of the protein; the change in form with acetylene confirms that acetylene binds to the MoFe protein. The EPR spectrum of Ca2 had features at g values of 2.04, 1.95 and 1.89 and an integrated intensity of 0.28 electron/molecule of protein. The MoFe protein and Fe protein of C. autotrophicum revealed no cold lability. The pH optima for C 2 H 2 , N 2 and H 3 O + reductions occurred between pH 7.1–7.3 with Bes buffer; in Pipes buffer the reduction of all three substrates was inhibited by 40%. At high molar ratios of Ca1 protein to Ca2 protein C 2 H 2 reduction suffered a lag phase before a linear rate occurred. The rate of N 2 reduction was linear but low whereas the rate of H 3 O + reduction under argon remained constant. H 3 O + reduction under N 2 (concomitant H 2 evolution) increased with excess Ca1. The results suggest different sites or forms of the enzyme for the three substrates at the nitrogenase protein and a second role for the Fe protein during N 2 reduction. Two ferredoxins were isolated with the optical properties of eight‐iron, eight‐sulfur proteins. They exhibited an absorption ratio A 390 / A 280 of 0.71 (Fd I) and 0.76 (Fd II) and peaks at 280 nm (Fd I), 285 nm (Fd II) and 390 nm and shoulders at 305 nm. The molecular weights were 12400 (Fd I) and 12800 (Fd II) in sodium dodecylsulfate gels and 12900 for a mixture of both proteins using thinlayer gel filtration. Fd I contained 10.0 mol Fe/mol and 9.4 mol S 2− /mol, Fd II 8.1 mol Fe/mol and 10.4 mol S 2− /mol, as determined colourimetrically. Using the atomic absorption method 7.9 mol Fe/mol protein and 8.4 mol Fe/mol protein were measured for Fd I and Fd II, respectively. Both proteins gave identical EPR spectra in the oxidised ( g = 2.00) and in the reduced state ( g = 2.07, 1.94, 1.88) which integrated to 0.12 electron per molecule of protein in both instances. Since the ferredoxins isolated from obligate aerobic nitrogen‐fixing bacteria such as Azotobacter vinelandii and Mycobacterium flavum exhibit paramagnetic features only in the oxidised state, the two ferredoxins of C. autotrophicum represent a new type of ferredoxin within this group.