The genes hoxF, -U, -Y, and -H which encode the four subunit polypeptides a, -y, 8, and 1 of the NADreducing hydrogenase (HoxS) ofAkaligenes eutrophus H16, were cloned, expressed in PseudomonasfacUis, and sequenced. On the basis of the nucleotide sequence, the predicted amino acid sequences, and the N-terminal amino acid sequences, it was concluded that the structural genes are tightly linked and presumably organized as an operon, denoted hoxS. Two pairs of -24 and -12 consensus sequences resembling RpoN-activatable promoters lie upstream of hoxF, the first of the four genes. Primer extension experiments indicate that the second promoter is responsible for hoxS transcription. hoxF and hoxU code for the flavin-containing dimer (a and y subunits) of HoxS which exhibits NADH:oxidoreductase activity. A putative flavin-binding region is discussed. The 26.0-kilodalton (kDa) y subunit contains two cysteine clusters which may participate in the coordination of two [4Fe-4S] centers. The genes hoxY and hoxH code for the small 22.9-kDa 8 subunit and the nickel-containing 54.8-kDa ,B subunit, respectively, of the hydrogenase dimer of HoxS. The latter dimer exhibits several conserved regions found in all nickel-containing hydrogenases. The roles of these regions in coordinating iron and nickel are discussed. Although the deduced amino acid sequences of the 8 and ,1 subunits share some conserved regions with the corresponding polypeptides of other [NiFe] hydrogenases, the overall amino acid homology is marginal. Nevertheless, significant sequence homology (35%) to the corresponding polypeptides of the soluble methylviologen-reducing hydrogenase of Methanobacterium thermoautotrophicum was found. Unlike the small subunits of the membrane-bound and soluble periplasmic hydrogenases, the HoxS protein does not appear to be synthesized with an N-terminal leader peptide.Alcaligenes eutrophus H16 is a gram-negative facultatively lithoautotrophic bacterium which can assimilate CO2 and utilize H2 as an energy source (reviewed in reference 5). Hydrogen oxidation is catalyzed by hydrogenases. These enzymes are found in phylogenetically diverse microorganisms, where they are responsible for both consumption and production of H2 (reviewed in reference 13). Two distinct hydrogenases have been purified and characterized from extracts of A. eutrophus (39, 44). These two enzymes differ in cellular localization, cofactor content, polypeptide composition, and apparent molecular weight. [NiFeSe]. An enzyme of this type has been described for Desulfovibrio and Methanococcus strains (reviewed in reference 13).The genes coding for the two hydrogenases of A. eutrophus H16 lie in a cluster of genes on a 450-kilobase-pair (kb) conjugative megaplasmid (15,24). Molecular cloning of megaplasmid DNA in Escherichia coli and screening of the resultant hybrid plasmids for complementation of hydrogenase-deficient mutants led to the identification of hydrogenase (hox) genes. Subsequent studies revealed that the two structural gene loci hoxS and hoxP coding for...

The cytoplasmic, tetrameric NAD-linked hydrogenase from Nocardia opaca lb can be separated in two dimeric substructures, an a~-dimer with NADH:electron acceptor oxidoreductase (diaphorase) activity and a ffb-dimer which displays hydrogenase activity with artificial electron carriers. These two dimers were preparatively isolated by a FPLC Mono Q procedure in the absence of nickel and at alkaline pH values. The hydrogenase-active /~b-dimer contained, as analyzed by inductively coupled plasma mass spectrometry (ICP-MS), 3.5-3.9 iron atoms and 1.3-1.7 nickel atoms per dimer molecule. EPR and Miissbauer spectra indicated the presence of a [4Fe-4S] cluster. This center turned out to be extremely labile towards oxidants. Oxidation led to irreversible convertion into a [3Fe-4S] form, thus representing an artifact and not a regulatory state of the cluster. The midpoint redox potential of the [4Fe-4S] cluster was determined to be -385 mV. Very weak EPR Ni signals of the /l~-dimer were detectable in the oxidized as well as in the reduced state. The diaphorase-active a~-dimer was free of nickel and the iron content corresponded to 11.2-12.8 Fe atoms per dimer molecule. From EPR and Miissbauer measurements it was concluded that this dimer contained two [4Fe-4S] clusters, one [2Fe-2S] and one [3Fe-4S] cluster. In accordance with the results obtained for the dimer proteins, for the whole enzyme an iron content of 15.8-16.2 atoms per enzyme molecule have been determined. EPR spectra and spectrum simulations of the native hydrogenase corroborate the cluster assignments of the two dimers: in total the enzyme contains one [2Fe-2S] cluster, one [3Fe-4S] cluster and three [4Fe-4S] clusters.

The cytoplasmic, NAD-linked hydrogenase of the Gram-positive hydrogen-oxidizing bacterium Nocardia opaca 1 b was compared with the analogous enzyme isolated from the Gram-negative bacterium Alcaligenes eutrophus H16. The hydrogenase of N. opaca 1b was purified by a new procedure applying chromatography on phenyl-Sepharose and DEAE-Sephacel with two columns in series. A homogeneous enzyme preparation with a specific activity of 74 pmol H2 oxidized . min-' . mg protein-' and a yield of 32% was isolated. The A. eutrophus enzyme was purified as previously published. Both enzymes are tetrameric proteins composed of four nonidentical subunits (a, / 3, y, 6). The four subunits of both of these enzymes were separated and isolated as single polypeptides by preparative polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.Immunological comparison of the four subunits of the Nocardia hydrogenase with those of the Alcaligenes enzyme showed that the a, p, y, and 6 subunits of one organism were serologically related to the analogous subunits of the other organism. Among themselves, the four subunits do not have any serological relationship. The eight individual polypeptides were also compared with respect to the NH2-terminal amino acid sequences determined by automated Edman degradation and to the amino acid compositions. Strong sequence similarities exist between the analogous subunits isolated from the two bacteria. Within the established N-terminal sequences the similarities between both a, fl, y and 6 subunits amount to 63%, 79%, 80% and 65%, respectively. No similarities exist between the different, non-analogous subunits a, /3, y and 6.Cytoplasmic, NAD-linked hydrogenases have been so far detected in only two groups of organisms, in the Gram-positive bacterium Nocardia opaca l b and in the Gram-negative bacterium Alcaligenes eutrophus H16 and closely related species of the same genus. In N. opaca and A . ruhlandii the NAD-linked hydrogenase is the only hydrogen-activating enzyme. A . eutrophus H16 contains in addition a membranebound hydrogenase which channels electrons directly into the electron transport chain [l]. The majority of the aerobic, autotrophic hydrogen-oxidizing bacteria as well as most aerobic nitrogen-fixing bacteria so far studied contain only a membrane-bound hydrogenase.The cytoplasmic, NAD-linked hydrogenases from N . opaca l b and A . eutrophus H16 have very similar structural properties. To date the most astonishing similarity of both enzymes is their composition. Both are tetramers composed of four non-identical subunits which in the case of Nocardia hydrogenase have M , of 64000 (a subunit), 56000 (p subunit), 31 000 (y subunit) and 27000 (6 subunit) and in the case of the soluble Alcaligenes hydrogenase M , of 63 000 (a subunit), Enzymes. Hydrogenase or H2 : NAD+ oxidoreductase (EC 1.12.1.2); hydrogenase or hydrogen : acceptor oxidoreductase (EC 1.12.-.-); Fa2,-reducing hydrogenase, (EC 1 56000 (p subunit), 30000 (y subunit) and 26000 (6 subunit)[2]. The composition of cof...

Potentiometric biosensors for the determination of organophosphorus (OP) insecticides were developed by applying either immobilized whole cells or cytoplasmic membrane fractions of wild-type Flavobacterium sp. on the surface of a glass pH electrode. The ability of Flavobacterium sp. to degrade OP compounds as sole carbon source was demonstrated for parathion with a degradation rate of almost 100% after 30 min and for chlorpyrifos of 33% after 48 h incubation. The products of hydrolysis of these compounds, p-nitrophenol and 3,5,6-trichloro-2-pyridinol, were accumulated in the medium and not used as substrates for growth by Flavobacterium sp. In the course of hydrolysis, which is catalyzed by organophosphorus hydrolase, two protons are released for each substrate molecule hydrolyzed. This stoichiometry forms the electrochemical basis of the potentiometric biosensors. Direct determination without previous extraction of OP was carried out in a stirred measuring cell with a pH electrode as transducer. Poly(carbamoyl sulfonate) (PCS) prepolymer, a hydrogel with good adhesive properties, was used for immobilization of whole cells and membrane-associated organophosphorus hydrolase. The sensor with cytoplasmic membrane fractions was superior to the one with whole cells and showed a linear range for paraoxon from 0.01 to 0.47 mM and 3 weeks' working stability.

A biosensor consisting of immobilized microbial cells and an oxygen electrode was used in a flow-through system as a microbial sensor flow injection analyzer (FIA). For different organic analytes, the metabolism of vital cells provides individual time-resolved responses with distinct time-dependent amplitudes. Chemometrical data analysis revealed that the individual responses are additive and depend linearly on single analyte concentrations. Based on these observations, simultaneous multicomponent analysis of organic mixtures was carried out in the FIA's time domain with analytical errors of less than 10%. For mixture analysis and monitoring in processes like enzymatic conversions, the described microbial sensor FIA ("dynamic microbial sensor") offers an alternative to expensive analytical equipment.