Hydrogenases that are composed of two dissimilar subunits have been identified as beterodimeric (NiFe) or (NiFeSe) hydrogenases. Hydrogenases having more than two subunits are designated as multimeric (NiFe) or (NiFeSe) hydrogenases. The multimeric hydrogenases can be further subdivided on the basis of the involvement of the unique electron acceptors, F420 and NAD + as the F420-(NiFe) hydzogenases and the NAD+-(NiFe) hydrogenase. This connotation reflects the molecular relationships within the gene family and accommodates a number of biochemical realities. NICKEL HYDROOENASE FAMILY

The genes encoding the two structural subunits of Escherichia coli hydrogenase 2 (HYD2) have been cloned and sequenced. They occur in an operon (hyb) which contains seven open reading frames. An hyb deletion mutant (strain AP3) failed to grow on dihydrogen-fumarate medium and also produced very low levels of HIYD1. All seven open reading frames are required for restoration of wild-type levels of active HYD2 in AP3.The hyb operon was mapped at 65 min on the E. coli chromosome.Under anaerobic growth conditions, Escherichia coli produces three different nickel-containing hydrogenases (3, 39). Hydrogenase 3 (HYD3) is part of the formate hydrogenlyase (FHL) complex and is responsible for formate-dependent dihydrogen (H2) evolution. The operon encoding HYD3 and other accessory electron transport components of the FHL complex, hyc, has been identified and is located at 58 min on the E. coli chromosome (7). The highly oxygen-labile nature of HYD3 has precluded detailed biochemical characterization. HYD2 is involved in H2 uptake and can be differentially induced to high levels when cells are grown in medium containing H2 as an electron donor and fumarate as an electron acceptor (3,23,25,39). An active component of HYD2 has been purified and shown to be a heterodimeric enzyme with a 58-kDa large subunit and a 30-kDa small subunit (4). Although mutants defective in H2 uptake have been described (23,25), detailed analysis of the operon encoding HYD2 has not been carried out. HYDl has also been purified and shown to consist of a large (60 kDa) subunit and a small (30 kDa) subunit (16,40). An active form of HYD1 containing only the large subunit has also been purified and characterized (1, 16). The operon encoding the two structural subunits of HYD1 (hya) contains a total of six genes and has been mapped at 22 min on the E. coli chromosome (30,31). The function of HYD1 is not understood, but it is believed to have a role in hydrogen cycling during fermentative growth. In addition to the operons coding for the structural components of the three hydrogenases, a fourth operon, hyp, located at 58 min, is essential for activity of all three hydrogenases (20,26,38). At least one of the genes in this operon (hypB) is involved in nickel metabolism, most probably via nickel insertion into apoenzyme (27).In this paper, we present the DNA sequence of the operon encoding HYD2 (hyb), which contains seven open reading frames (ORFs). Cassette mutagenesis of the hyb operon on the chromosome resulted in a total loss of HYD2 expression and activity, as well as in significant reduction in HYD1 activity. MATERIALS AND METHODSBacterial strains. All bacterial strains used were E. coli K-12 derivatives and are listed in Table 1 [pH 7.0]), resuspended in the same buffer to an optical density of 0.5 at 600 nm, and used for whole-cell enzyme assays. Cell extracts were prepared by sonicating cell suspensions on ice with a model W385 sonicator (Heat Systems) for 20 5-s bursts. Triton X-100 was added to a final concentration of 2% (vol/vol), when req...

No abstract

The hydrogenase from Desulfovibrio baculatus (DSM 1743) was purified from each of three different fractions: soluble periplasmic (wash), soluble cytoplasmic (cell disruption) and membrane-bound (detergent solubilization). Plasma-emission metal analysis detected in all three fractions the presence of iron plus nickel and selenium in equimolecular amounts. These hydrogenases were shown to be composed of two non-identical subunits and were distinct with respect to their spectroscopic properties. The EPR spectra of the native (as isolated) enzymes showed very weak isotropic signals centered around g x 2.0 when observed at low temperature (below 20 K). The periplasmic and membrane-bound enzymes also presented additional EPR signals, observable up to 77 K, with g greater than 2.0 and assigned to nickel(II1). The periplasmic hydrogenase exhibited EPR features at 2.20, 2.06 and 2.0. The signals observed in the membrane-bound preparations could be decomposed into two sets with g at 2.34, 2.16 and x 2.0 (component I) and at 2.33, 2.24, and x 2.0 (component 11). In the reduced state, after exposure to an Hz atmosphere, all the hydrogenase fractions gave identical EPR spectra. EPR studies, performed at different temperatures and microwave powers, and in samples partially and fully reduced (under hydrogen or dithionite), allowed the identification of two different iron-sulfur centers : center I (2.03, 1.89 and 1.86) detectable below 10 K, and center I1 (2.06, 1.95 and 1.88) which was easily saturated at low temperatures. Additional EPR signals due to transient nickel species were detected with g greater than 2.0, and a rhombic EPR signal at 77 K developed at g 2.20, 2.16 and 2.0. This EPR signal is reminiscent of the Ni-signal C (g at 2.19, 2.14 and 2.02) observed in intermediate redox states of the well characterized Desulfovibrio gigas hydrogenase (Teixeira et al. (1985) J. Biol. Chem. 260, 89421. During the course of a redox titration at pH 7.6 using Hz gas as reductant, this signal attained a maximal intensity around -320 mV. Low-temperature studies of samples at redox states where this rhombic signal develops (10 K or lower) revealed the presence of a fastrelaxing complex EPR signal with g at 2.25, 2.22, 2.15, 2.12, 2.10 and broad components at higher field. The soluble hydrogenase fractions did not show a time-dependent activation but the membrane-bound form required such a step in order to express full activity. This indicates that the redox state of the isolated enzyme is important for the full expression of enzymatic activity. The catalytic properties were also followed by the proton-deuterium exchange reaction. The isolated hydrogenases produced Hz/HD ratios higher than those observed for nonselenium-containing hydrogenases.The enzyme responsible for the biological activation of H2, termed hydrogenase [l, 21, has a central role in many relevant anaerobic processes where molecular hydrogen is oxidized or evolved. Also, molecular hydrogen, via the hydrogenase system, is a link between different bacterial consortia w...

Below 30 K, oxidized Desulfovibrio gigas hydrogenase presents an intense electron paramagnetic resonance (EPR) signal centered at g=2.02, typical of an iron-sulfur center. In addition a rhombic EPR signal, attributed to Ni(1II) species, is also observed [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. Biop!?j>s. Rcs. Commun. 106,[610][611][612][613][614][615][616] and Cammack, R., Patil, D., Aguirre, R., and Hatchikian, E. C., (1982) FEBS Lett. 142, 289-2921, At higher temperatures (77 K) the iron-sulfur EPR signal is broader and all the EPR features of the rhombic nickel signal can easily be observed. We have now obtained additional information concerning the redox properties of these EPR active centers, using an EPR redox titration method in the presence of dye mediators at pH = 8.5, The mid-point potential was determined to be -70 mV for the Fe,S cluster and -220 mV for the Ni center. Intermediate oxidation states were obtained upon partial reduction with either dithionite or hydrogen. Although upon dithionite reduction the centers are reduced in the order of decreasing mid-point reduction potentials, under a hydrogen atmosphere the nickel center reduces preferentially. This suggests a catalytic involvement of the nickel redox center in the binding of hydrogen. Preliminary Mossbauer studies on Desulfovibrio gigas hydrogenase reveal the presence of a paramagnetic 3 Fe center and two 4 Fe centers. The 3 Fe center is responsible for the g= 2.02 EPR signal but the two 4 Fe centers have been so far undetectable by EPR.Iron-sulfur centers have been implicated in the simplest known oxidation-reduction process which is mediated by hydrogenase, i.e. the reduction of the proton [I].Flavins have also been indicated as prosthetic groups in a few cases [2]. Nickel has been shown to be required for the biosynthesis of hydrogenase [3] and found in the hydrogenase of Methanobacterium thermoautotrophicum [4].Recently the presence of Ni in several hydrogenases was firmly established by EPR studies of 61Ni-enriched samples. A rhombic EPR signal with g values at 2.30, 2.23 and 2.02 was previously observed in oxidized membranes of Methanobacterium bryantii [ 5 ] . This rhombic signal was later proven to originate from Ni through the observation of hyperfine structure on the EPR spectrum of samples prepared from cells grown in 61Ni-enriched culture medium [6]. The same type of Ni signal was then observed in hydrogenases from Mb. thermoautotrophicum [7], Desulfovibrio gigas [8] and Desulfovibrio desulfuricans (strain 27774) [9].Desulfovibrio gigas hydrogenase was recently purified by a new improved method and a nickel content of approximately 0.7 mol Nii89 kDa was found IS]. In the oxidized state (native form) superimposed with an intense narrow EPR signal centered around g = 2.02, a rhombic signal was also detected with g values of 2.31,2.23, and around 2.00. The g values, line shape and saturation characteristics were si...

flavoprotein of high reducing potential (Elo approximately-0.34 v.). Thioctic reductase is present also in spinach chloroplasts. Note added in proof.-Since this paper was submitted for publication, Massey [Biochim. Biophys. Acta, 32, 286, (1959)] has provided additional evidence that diaphorase is a component of the KG dehydrogenase complex.

This paper presents a Q-band ENDOR study of the nickel site of the as-isolated (Ni-A), H2-reduced (Ni-C), and reoxidized (Ni-A/Ni-B) states of Desulfovibrio gigas hydrogenase. Through proton and deuteron ENDOR measurements we detect and characterize the possible products of heterolytic cleavage of H2, namely two distinct types of exchangeable protons, bound to the Ni-C site. One proton, H(l), has a hyperfine coupling, AH( 1) = 16.8 MHz and appears to interact directly with Ni-C. The other proton, H(2), has AH(2) =» 4.4 MHz and could be associated with H20 or OH" bound to nickel. We discuss possible binding modes for H(l) and H(2). One type of exchangeable deuteron(s), D(2), associated with the Ni-C center remains associated with the Ni-B center after oxidation of the Ni-C. In addition we confirm that the Ni-A site is inaccessible to solvent protons.

No abstract