Hans Wassink

Novel structure and redox chemistry of the prosthetic groups of the iron-sulfur flavoprotein sulfide dehydrogenase from Pyrococcus furiosus; evidence for a [2Fe-2S] cluster with Asp(Cys) (3) ligands

2]2 − and its ability to bind to biological macromolecules should not be overlooked, and may artificially trigger/accelerate Cu(II) reduction.

Formation and characterization of a transition state complex of Azotobacter vinelandii nitrogenase

A stable complex is formed between the nitrogenase proteins of Azotobacter vinelandii, aluminium fluoride and MgADP. All nitrogenase activities are inhibited. The complex formation was found to be reversible. An incubation at 50°C recovers nitrogenase activity. The complex has been characterized with respect to protein and nucleotide composition and redox state of the metal‐sulphur clusters. Based on the inhibition by aluminium fluoride together with MgADP, it is proposed that a stable transition state complex of nitrogenase is isolated.

Bioreduction of carboxylic acids by Pyrococcus furiosus in batch cultures

The reduction of aromatic and aliphatic (di)carboxylic acids to their corresponding aldehydes and alcohols by the hyperthermophilic organism Pyrococcus furiosus was investigated. The reduction was performed with P. furiosus cells growing in the presence of 1 mM acid with starch as a carbon and energy source at 90°C. The aromatic acids t-cinnamic and 3-phenylpropionic acid were reduced to their corresponding alcohols with the highest yields in the described batch cultures: 67 and 69%, respectively. The aliphatic acid reduced with the highest yield was hexanoic acid (yield: 38%). No aldehydes were detected during the reduction of acids, indicating that the reduction of aldehydes to alcohols is faster than the reduction of acids to aldehydes. Some aldehydes were both reduced to the corresponding alcohol and oxidized to the corresponding acid. Besides reduction of the unsaturated t-cinnamaldehyde to t-cinnamyl alcohol (63%), the double bond of t-cinnamaldehyde was also reduced by P. furiosus.

Pre-steady-state Kinetics of Nitrogenase from Azotobacter vinelandii EVIDENCE FOR AN ATP-INDUCED CONFORMATIONAL CHANGE OF THE NITROGENASE COMPLEX AS PART OF THE REACTION MECHANISM

The pre-steady-state electron transfer reactions of nitrogenase from Azotobacter vinelandii have been studied by stopped-flow spectrophotometry. With reduced nitrogenase proteins after the initial absorbance increase at 430 nm (which is associated with electron transfer from the Fe protein to the MoFe protein and is complete in 50 ms) the absorbance decreases, which, dependent on the ratio [Av2]/[Av1], is followed by an increase of the absorbance. The mixing of reductant-free nitrogenase proteins with MgATP leads after 20 ms to a decrease of the absorbance, which could be fitted (from 0.05 to 1 s) with a single exponential decay with a rate constant kobs = 6.6 ± 0.8 s−1. This reaction of nitrogenase was measured at different wavelengths. The data indicate the formation of a species with a blue shift of the absorbance of metal-sulfur clusters of nitrogenase from 430 to 360 nm. The absorbance decrease at 430 nm observed (after 50 ms) in the case of the reduced nitrogenase proteins could only be simulated well if, after the initial electron transfer from the Fe protein to the MoFe protein and before dissociation of the nitrogenase complex, an additional reaction was assumed. The rate constant of this reaction was of the same order as the rate constant of the MgATP-dependent pre-steady-state proton production by nitrogenase from A. vinelandii: kobs = 14 ± 4 s−1 with reduced nitrogenase proteins and kobs = 6 ± 2 s−1 with dithionite-free nitrogenase proteins (Duyvis, M. G., Wassink, H., and Haaker, H. (1994) Eur. J. Biochem. 225, 881-890). It is proposed that in the presence and absence of reductant, the observed absorbance decrease at 430 nm of nitrogenase is caused by a change of the conformation of the nitrogenase complex, as a consequence of hydrolysis of MgATP.

Evidence for multiple steps in the pre-steady-state electron transfer reaction of nitrogenase from Azotobacter vinelandii.

Abstract The effect of the NaCl concentration and the reaction temperature on the MgATP-dependent pre-steady-state electron transfer reaction (from the Fe protein to the MoFe protein) of nitrogenase from Azotobacter vinelandii was studied by stopped-flow spectrophotometry and rapid-freeze EPR spectroscopy. Besides lowering the reaction temperature, also the addition of NaCl decreased the observed rate constant and the amplitude of the absorbance increase (at 430 nm) which accompanies pre-steady-state electron transfer. The diminished absorbance increase observed at 5°C (without NaCl) can be explained by assuming reversible electron transfer, which was revealed by rapid-freeze EPR experiments that indicated an incomplete reduction of the FeMo cofactor. This was not the case with the salt-induced decrease of the amplitude of the stopped-flow signal: the observed absorbance amplitude of the electron transfer reaction predicted only 35% reduction of the MoFe protein, whereas rapid-freeze EPR showed 80% reduction of the FeMo cofactor. In the presence of salt, the kinetics of the reduction of the FeMo cofactor showed a lag period which was not observed in the absorbance changes. It is proposed that the pre-steady-state electron transfer reaction is not a single reaction but consists of two steps: electron transfer from the Fe protein to a still unidentified site on the MoFe protein, followed by the reduction of the FeMo cofactor. The consequences of our finding that the pre-steady-state FeMo cofactor reduction does not correlate with the amplitude and kinetics of the pre-steady-state absorbance increase will be discussed with respect to the present model of the kinetic cycle of nitrogenase.

Iron-sulfide content and ATP binding properties of nitrogenase component II from Azotobacter vinelandii

Nitrogenase is the enzyme system that catalyses the reduction of N2 to ammonia. Nitrogenase consists of two separable proteins. A tetrameric MoFe-protein and a dimeric Fe-protein (Mortenson, Thorneley, 1979). Electrons are donated to the Fe-protein and pass to the MoFe- protein ATP-dependently (Mortenson, Thorneley, 1979; Hageman, Burris, 1980). It is generally accepted that the Fe-protein of any nitrogenase enzyme complex has one [4Fe-4S] cluster (Mortenson, Thorneley, 1979; Eady, 1980; Orme-Johnson, Munck, 1980). This is based upon iron and sulfide determinations of various Fe-proteins of nitrogenase isolated from different bacteria (Eady, 1980). Cluster extrusion experiments indicate that the iron and sulfide in the Cp2 preparations are quantitatively recovered as a similar product as found with ferredoxin, i. e. a [4Fe-4S] cluster (Gillum et al. 1977). The Fe-protein from C. pasteurianum behaves like a one electron donor/ acceptor upon redox titrations in EPR experiments (Zumft et. al, 1974) and in colorimetric measurements (Ljones, Burris, 1978). However, there are reports that the Fe-protein from Azotobacter can accommodate two electrons (Thorneley et. al., 1976, Braaksma et al., 1982). Lowe (1978) proposed that in view of the anisotropic linewidth and the low integrated EPR signal intensities, a second rapidly relaxing paramagnetic center is present in the protein. In this article we present data that highly-active Fe protein can be isolated from A. vinelandii that contains 8 iron and 8 sulfide atoms per dimer of 63 kDa.