Electron paramagnetic resonance and other properties of hydrogenases isolated from Desulfovibrio vulgaris (strain Hildenborough) and Megasphaera elsdenii

Abstract

The hydrogenases of DesuEfovibrio vulgaris and Megasphaera elsdenii are compared with respect to some of their physical properties. In addition to Fe the only metal ions that are present in significant amounts are Ni and Cu. From cluster extrusion experiments it follows that the D. vulgaris enzyme contains three 4 Fe-4 S clusters, while M . elsdenii hydrogenase only releases part of its Fe-S clusters. The resting D . vulgarisenzyme shows only a small 3 Fe-xS type of EPR signal (maximum 5 %electronequivalent). This amount can beincreased to approximately25 %by treatment with ferricyanide, with a concomitant large decrease in activity. The M. elsdenii enzyme shows in its oxidized state a normal Hipip (high-potential iron-sulphur protein) type of EPR spectrum. After a reduction/oxidation cycle the D. vulgaris enzyme also shows a weak Hipip type of EPR spectrum. In the reduced state both enzymes show complex spectra. By integration of those spectra it is shown that 1.5 electron equivalents are present. The complex spectra do not arise from nuclear hyperfine interactions but are partially due to electron spin interactions. It is proposed that the spectrum of reduced D. vulgaris hydrogenase consists of a sum of three different ferredoxin-like spectra.

The relationship between activity and the axial g=2.06 EPR signal induced by CO in the periplasmic (Fe) hydrogenase from Desulfovibrio vulgaris

“…The native (Fe) hydrogenase shows a weak g= 2.02 isotropic EPR signal which integrates to approx. 0.02 spin/molecule and has been reported previously [3,4]. This signal is characteristic of a three-iron center but, because of its low spin quantitation, does not appear to be of catalytic signifisignificance.…”

“…56 kDa and is composed of two subunits (46 kDa, 10.5 kDa) [1,2]. Spectroscopic and cluster-extrusion data indicate the prosthetic groups to be two ferredoxin-type [4Fe-4S] clusters and an iron cluster with abnormal spectroscopic properties [3,4]; however, the total iron content has been reported to vary between 11 and 16 atoms per molecule [3,5]. The unique sensitivity of the hydrogenase activity found in D. vulgaris to CO was first recognized by Krasna and Rittenberg [6] but its significance was not appreciated until the presence of multiple hydrogenases with different biochemical properties in this bacterium was reported [7].…”

Purification and properties of the soluble hydrogenase from Desulfovibrio desulfuricans (strain Norway 4)

“…Signals a t g = 2.02 are observed in most hydrogenase [27], although Albracht et al [24] reported that such signals were absent from hydrogenase of Mrthunobucterium therrnoautotrophicum, Marburg strain. The [3Fe-sS] signal in Desul/i)iihrio iwlguris (Hildenborough) hydrogenase was reported to be only 5% electron equivalent [28]. Signals due to nickel(II1) have been observed in most but not all nickel-containing hydrogenases [3].…”

Putative signal peptide on the small subunit of the periplasmic hydrogenase from Desulfovibrio vulgaris

“…The NH2-terminal amino acid sequence of the large subunit is colinear with the AUG translational start of the gene, suggesting the absence of a signal peptide and leading to speculation that the gene might code for a cytoplasmic hydrogenase. A high degree of homology between the NH2-terminal region of the large subunit and the amino acid sequences of the eight iron ferredoxins substantiates the biochemical information, indicating that two of the three iron clusters are of the ferredoxin type (7,11). As 10 of the remaining 11 cysteine residues were located on the large subunit, it was suggested that the third Fe4S4 cluster is also located on the large subunit; however, the possible involvement of the single cysteinyl residue on the small subunit as a ligand for an iron-sulfur cluster could not be eliminated.…”