B H Huynh

The presence of redox-sensitive nickel in the periplasmic hydrogenase from Desulfovibriogigas

Abstract A new and improved method for the purification of the periplasmic hydrogenase from Desulfovibrio gigas is described. This preparation of hydrogenase was found to contain 0.64 g atom of nickel per mole of protein. In the oxidized state, the hydrogenase exhibited an isotropic signal at g = 2.02 and a characteristic Ni(III) signal with g-values at 2.31, 2.20 and ∼2.0. The EPR spectrum of the reduced enzyme consisted of multiple species. One set of g-values are determined as 2.17, 2.08 and 2.04. The other minor species exhibited a resonance at g = 2.28. On partial reoxidation of the hydrogenase, the initial Ni(III) signals reappeared along with additional signals attributed to multiple Ni(III) species. It is proposed that Ni is an important functional unit in this hydrogenase.

A Proposed Role for the Azotobacter vinelandii NfuA Protein as an Intermediate Iron-Sulfur Cluster Carrier

Iron-sulfur clusters ([Fe-S] clusters) are assembled on molecular scaffolds and subsequently used for maturation of proteins that require [Fe-S] clusters for their functions. Previous studies have shown that Azotobacter vinelandii produces at least two [Fe-S] cluster assembly scaffolds: NifU, required for the maturation of nitrogenase, and IscU, required for the general maturation of other [Fe-S] proteins. A. vinelandii also encodes a protein designated NfuA, which shares amino acid sequence similarity with the C-terminal region of NifU. The activity of aconitase, a [4Fe-4S] cluster-containing enzyme, is markedly diminished in a strain containing an inactivated nfuA gene. This inactivation also results in a null-growth phenotype when the strain is cultivated under elevated oxygen concentrations. NifU has a limited ability to serve the function of NfuA, as its expression at high levels corrects the defect of the nfuA-disrupted strain. Spectroscopic and analytical studies indicate that one [4Fe-4S] cluster can be assembled in vitro within a dimeric form of NfuA. The resultant [4Fe-4S] cluster-loaded form of NfuA is competent for rapid in vitro activation of apo-aconitase. Based on these results a model is proposed where NfuA could represent a class of intermediate [Fe-S] cluster carriers involved in [Fe-S] protein maturation.

Unambiguous identification of the nickel EPR signal in 61Ni-enriched Desulfovibrio gigas hydrogenase

Summary A highly active hydrogenase from Desulfovibrio gigas (sp. act. 440 μmoles H 2 evolved/min. mg) was purified from cells grown in 61 Ni enriched medium. The nuclear spin (I = 3/2) of 61 Ni induces hyperfine structure in the EPR spectra of purified hydrogenase, unequivocally identifying the previously observed signal as a Ni(III) species (LeGall, J., Ljungdahl, P., Moura, I., Peck, H.D. Jr., Xavier, A.V., Moura, J.J.G., Teixeira, M., Huynh, B.H. and DerVartanian, D.V., (1982) Biochem. Biophys. Res. Commun. 106 , 610–616). Samples reduced under hydrogen also show hyperfine structure suggesting the presence of a transient Ni(III) species in the reduced active state of the enzyme.

Conversion of [3 Fe3 S] into [4 Fe4 S] clusters in a Desulfovibrio gigas ferredoxin and isotopic labeling of iron-sulfur cluster subsites

T. A. KENT+, I. MOURA+**, J. J. G. MOURA+

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

*, J. D. LIPSCOMB, B. H. HUYNI-I+pt , J. LeGALL*, A. V. XAVIER+>* and E. MUNCK+ Department of Biochemistry, University of Minnesota, Minneapolis, MN 55455, fDepartment of Physics, Emory University, Atlanta, GA 30322, *Department of Biochemistry, University of Georgia, Athens, GA 30602, +Gray Freshwater Biological Institute, University of Minnesota, Navarre, MN 55392, USA and *Centro de Quimica Estrutural das Universidades de Lisboa IST, 1000 Lisbon, Portugal

Characterization of two dissimilatory sulfite reductases from sulfate-reducing bacteria

The effect of exposure to carbon monoxide on the activity of the (Fe) hydrogenase from Desulfovibrio vulgaris has been determined. Concentrations of carbon monoxide which completely inhibit hydrogenase activity and induce formation of the axial g=2.06 EPR signal up to 0.8 spin/molecule do not cause irreversible inhibition of the (Fe) hydrogenase.

Mössbauer and EPR evidence for nickel and 3Fe cluster in the hydrogenases of D. desulfuricans and D. gigas

Mössbauer, EPR, and biochemical techniques were used to characterize two dissimilatory sulfite reductases: desulforubidin fromDesulfovibrio baculatus strain DSM 1743 and desulfoviridin fromDesulfovibrio gigas. For each molecule of desulforubidin, there are two sirohemes and four [4Fe−4S] clusters. The [4Fe−4S] clusters are in the diamagnetic 2+ oxidation state. The sirohemes are high-spin ferric (S=5/2) and each siroheme is exchanged-coupled to a [4Fe−4S]2+ cluster. Such an exchange-coupled siroheme-[4Fe−4S] unit has also been found in the assimilatory sulfite reductase fromEscherichia coli/1/ and in a low-molecular weight sulfite reductase fromDesulfovibrio vulgaris/2/. For each molecule of defulfoviridin, there are two tetrahydroporphyrin groups and four [4Fe−4S]2+ clusters. To our surprise, we discovered that about 80% of the tetrahydroporphyrin groups, however, do not bind iron.

The three classes of hydrogenases from sulfate-reducing bacteria of the genus Desulfovibrio

Abstract Hydrogenases from Desulfovibrio desulfuricans (27774) and from Desulfovibrio gigas have been studied by EPR and Mossbauer spectroscopy. As isolated, hydrogenase from D. gigas exhibits an ‘isotropic’ EPR signal at g = 2.02 and a rhombic EPR signal at g = 2.31, 2.20 and 2.02. Isotopic substitution of 61Ni(I = 3 2 proves that the rhombic signal is due to Ni. A similar EPR spectrum was observed for D. desulfuricans hydrogenase, indicating that both enzymes contain an EPR active nickel center. Combining the Mossbauer and EPR data, the isotropic g = 2.02 EPR signal was shown to originate from a 3Fe cluster which may have oxygenous or nitrogenous ligands. In addition, the Mossbauer data also revealed two [4Feue5f82S] clusters in each molecule of hydrogenase.