Surprisingly uninhibited: The inhibition of hydrogenases by oxygen is intensely studied because this is the main obstacle to using these enzymes in biofuel cells. The hydrogenase from Clostridium acetobutylicum (see structure) was found to react surprisingly slowly with O2. The inhibition mechanism was elucidated and the kinetics were quantitatively defined. This is a prerequisite for improving the enzyme further by genetic engineering and for assessing its potential in technological devices.

NifB-co, a Fe-S cluster produced by the enzyme NifB, is an intermediate on the biosynthetic pathway for the iron molybdenum cofactor (FeMo-co) of nitrogenase. We have used Fe K-edge extended xray absorption fine structure (EXAFS) spectroscopy together with 57 Fe nuclear resonance vibrational spectroscopy (NRVS) to probe the structure of NifB-co while bound to the NifX protein from Azotobacter vinelandii. EXAFS analysis of the NifX:NifB-co complex yields Fe-S distances of 2.26 Å and Fe-Fe distances of 2.66 Å and 3.74 Å. Search profile analyses reveals the presence of a single Fe-N (or C, O) interaction at 2.04 Å, implying that the interstitial light atom proposed to be present in FeMo-co is already inserted into NifB-co. NRVS reveals strong bands from Fe-S bending and stretching modes peaking around 270, 315, 385, and 408 cm −1 . Additional intensity at ~185 -200 cm −1 is interpreted as a set of cluster 'breathing' modes similar to those seen for the FeMo-cofactor. The strength and location of these modes also strongly suggest that the FeMo-co interstitial light atom seen in the crystal structure is already in place in NifB-co. Both the EXAFS and NRVS data are best simulated using a Fe 6 S 9 X trigonal prism structure analogous to the 6Fe core of FeMo-co, although a 7Fe structure made by capping one trigonal 3S terminus with Fe cannot be ruled out. This implies that the interstitial light atom is already present at an early stage in FeMo-co biosynthesis prior to the incorporation of Mo and R-homocitrate.

In Clostridium acetobutylicum, [FeFe]-hydrogenase is involved in hydrogen production in vivo by transferring electrons from physiological electron donors, ferredoxin and flavodoxin, to protons. In this report, by modifications of the purification procedure, the specific activity of the enzyme has been improved and its complete catalytic profile in hydrogen evolution, hydrogen uptake, proton/deuterium exchange and para-H2/ortho-H2 conversion has been determined. The major ferredoxin expressed in the solvent-producing C. acetobutylicum cells was purified and identified as encoded by ORF CAC0303. Clostridium acetobutylicum recombinant holoflavodoxin CAC0587 was also purified. The kinetic parameters of C. acetobutylicum [FeFe]-hydrogenase for both physiological partners, ferredoxin CAC0303 and flavodoxin CAC0587, are reported for hydrogen uptake and hydrogen evolution activities.