Homologous and heterologous overexpression in Clostridium acetobutylicum and characterization of purified clostridial and algal Fe-only hydrogenases with high specific activities.

Clostridium acetobutylicum ATCC 824 was selected for the homologous overexpression of its Fe-only hydrogenase and for the heterologous expressions of the Chlamydomonas reinhardtii and Scenedesmus obliquus HydA1 Fe-only hydrogenases. The three Strep tag II-tagged Fe-only hydrogenases were isolated with high specific activities by two-step column chromatography. The purified algal hydrogenases evolve hydrogen with rates of around 700 micromol H(2) min(-1) mg(-1), while HydA from C. acetobutylicum (HydA(Ca)) shows the highest activity (5,522 micromol H(2) min(-1) mg(-1)) in the direction of hydrogen uptake. Further, kinetic parameters and substrate specificity were reported. An electron paramagnetic resonance (EPR) analysis of the thionin-oxidized HydA(Ca) protein indicates a characteristic rhombic EPR signal that is typical for the oxidized H cluster of Fe-only hydrogenases.

Localization of a substrate binding site on the FeMo-cofactor in nitrogenase: trapping propargyl alcohol with an alpha-70-substituted MoFe protein.

Substitution of the MoFe protein alpha-70(Val) residue with Ala or Gly expands the substrate range of nitrogenase, allowing the reduction of larger alkynes, including propargyl alcohol (HC[triple bond]CCH(2)OH). Herein, we report characterization of the alpha-70(Val)(-->)(Ala) MoFe protein with propargyl alcohol trapped at the active site. The alpha-70(Ala) variant MoFe protein was rapidly frozen during reduction of propargyl alcohol, resulting in the conversion of the resting-state FeMo-cofactor EPR signal (S = 3/2 and g = [4.41, 3.60, 2.00]) to a new state (S = 1/2 and g = [2.123, 1.998, 1.986]). This EPR signal of the new state increased in intensity with increasing propargyl alcohol concentration, consistent with the binding of a single substrate. The EPR signal of the propargyl alcohol state showed temperature and microwave power dependencies markedly different from those of the classic FeMo-cofactor EPR signal, consistent with the difference in spin. The new state is analogous to that induced by the binding of the inhibitor CO ("lo CO" state) to FeMo-cofactor in the wild-type MoFe protein. The (13)C ENDOR spectrum of the alpha-70(Ala) MoFe protein with trapped (13)C-labeled propargyl alcohol exhibited three well-resolved (13)C doublets centered at the (13)C Larmor frequency with isotropic hyperfine couplings of approximately 3.2, approximately 1.4, and approximately 0.7 MHz, indicating that the alcohol (or a fragment) is coordinated to the cofactor. The results presented here localize the binding site of propargyl alcohol to one [4Fe-4S] face of FeMo-cofactor and indicate roles for the alpha-70(Val) residue in controlling FeMo-cofactor reactivity.

The interstitial atom of the nitrogenase FeMo-cofactor: ENDOR and ESEEM show it is not an exchangeable nitrogen.

A recent high-resolution X-ray crystallographic study (1.16 A) of the Azotobacter vinelandii nitrogenase MoFe protein revealed a previously undetected electron density associated with the active site FeMo-cofactor. The density is located inside the cluster at the center of the "trigonal prism" of six irons and is assigned to a species "X". The identity of species X was not resolved, although the electron density is consistent with a single N, O, or C atom. One proposal is that X is an N atom that derives from and exchanges with N from N2 during catalysis. In the present study, we have examined this possibility by employing 14N and 15N isotopes of N2 along with ENDOR and ESEEM spectroscopies. The WT MoFe protein and alpha-359Arg-->Lys and alpha-381Phe-->Leu variants were allowed to turn over in the presence of 14N2 or 15N2, and then were examined as resting enzymes by ENDOR and ESEEM at X- and Q-bands to look for all 14N and 15N signals coupled to the electron spin of the FeMo-cofactor and to determine if any exchanged during turnover. We have found five peaks in Q-band pulsed ENDOR spectra that appear to arise not only from previously reported N1/N2, which give rise to the ESEEM, but also from one or two additional coupled nitrogens. None of the ENDOR and ESEEM signals vanish or are altered by catalytic turnover with 15N2, and no new 15N signal is detected, leading to the conclusion that if species X is a nitrogen atom, it does not exchange during dinitrogen reduction.