The nucleotide sequence has been determined for a twelve-gene operon of Escherichia coli designated the hyf operon (hyfASCD€FGH/R-focB). The hyf operon is located a t 5508-56.0 min and encodes a putative nine-subunit hydrogenase complex (hydrogenase four or Hyf), a potential formate-and 6%-dependent transcriptional activator, HyfR (related to FhIA), and a possible formate transporter, FocB (related to FocA). Five of the nine Hyf-complex subunits are related to subunits of both the E. coli hydrogenase-3 complex (Hyc) and the proton-translocating NADH : quinone oxidoreductases (complex I and Nuo), whereas two Hyf subunits are related solely to NADH : quinone oxidoreductase subunits. The Hyf components include a predicted 523 residue [Ni-Fe] hydrogenase (large subunit) with an N-terminus (residues homologous to the 30 kDa or NuoC subunit of complex 1. It is proposed that Hyf, in conjunction with formate dehydrogenase H (Fdh-H), forms a hitherto unrecognized respiration-linked proton-translocating formate hydrogenlyase (FHL-2). It is likely that HyfR acts as a formate-dependent regulator of the hyf operon and that FocB provides the Hyf complex with external formate as substrate.

The FNR protein of Escherichia coli is a redox-responsive transcription regulator that activates and represses a family of genes required for anaerobic and aerobic metabolism. Reconstitution of wild-type FNR by anaerobic treatment with ferrous ions, cysteine and the NifS protein of Azotobacter vinelandii leads to the incorporation of two [4Fe-4S]2+ clusters per FNR dimer. The UV-visible spectrum of reconstituted FNR has a broad absorbance at 420 nm. The clusters are EPR silent under anaerobic conditions but are degraded to [3Fe-4S]+ by limited oxidation with air, and completely lost on prolonged air exposure. The association of FNR with the iron-sulphur clusters is confirmed by CD spectroscopy. Incorporation of the [4Fe-4S]2+ clusters increases site-specific DNA binding about 7-fold compared with apo-FNR. Anaerobic transcription activation and repression in vitro likewise depends on the presence of the iron-sulphur cluster, and its inactivation under aerobic conditions provides a demonstration in vitro of the FNR-mediated aerobic-anaerobic transcriptional switch.

SUMMARYMutants of Escherichia coli KI 2 strain WGAS-GF+/LF+ were selected for their inability to use fumarate as terminal electron acceptor for supporting growth on glycerol or lactate in an atmosphere of H, plus 5 % COz. Eighty-three mutants were grouped into seven different categories according to their ability to grow on different media and their ability to produce gas during glucose fermentation. Enzymological and genetic studies indicated that the major class (type I), representing nearly 70 % of the isolates, lacked fumarate reductase and corresponded to thefrdA mutants studied previously (Spencer & Guest, 1973, 1974. Members of a second class (type 11) were phenotypically similar to men mutants, blocked in menaquinone biosynthesis. They differed from menA mutants in having lesions in the 4 to 51 min region of the chromosome rather than at 87 min. It was concluded that fumarate reductase and menaquinone are essential for anaerobic growth when fumarate serves as electron acceptor but not when nitrate performs this function. Fumarate reductase and menaquinone are also essential for H,-dependent growth on fumarate. Type 111 mutants, originallyfrdl?, were designated fnr because they were defective in fumarate and nitrate reduction and impaired in their ability to produce gas. The fnr gene was located at 28-5 min by its cotransducibility withpyrF(5-7 to 9.2%) and trpA (2-7 to 5'7%) and the gene order fnr-qmeA-pyrF-tpA was established. It was not possible to assign specific metabolic lesions to the fnr mutants nor to the remaining classes, which all exhibited pleiotropic phenotypes. Nevertheless, the results demonstrate that functional or organizational relationships exist between the fumarate reductase system, nitrate reduction and hydrogen production.

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The crystal structure of the recombinant thiamin diphosphate-dependent E1 component from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc) has been determined at a resolution of 1.85 A. The E. coli PDHc E1 component E1p is a homodimeric enzyme and crystallizes with an intact dimer in an asymmetric unit. Each E1p subunit consists of three domains: N-terminal, middle, and C-terminal, with all having alpha/beta folds. The functional dimer contains two catalytic centers located at the interface between subunits. The ThDP cofactors are bound in the "V" conformation in clefts between the two subunits (binding involves the N-terminal and middle domains), and there is a common ThDP binding fold. The cofactors are completely buried, as only the C2 atoms are accessible from solution through the active site clefts. Significant structural differences are observed between individual domains of E1p relative to heterotetrameric multienzyme complex E1 components operating on branched chain substrates. These differences may be responsible for reported alternative E1p binding modes to E2 components within the respective complexes. This paper represents the first structural example of a functional pyruvate dehydrogenase E1p component from any species. It also provides the first representative example for the entire family of homodimeric (alpha2) E1 multienzyme complex components, and should serve as a model for this class of enzymes.