Wolinella succinogenes performs oxidative phosphorylation with fumarate instead of O2 as terminal electron acceptor and H2 or formate as electron donors. Fumarate reduction by these donors ('fumarate respiration') is catalyzed by an electron transport chain in the bacterial membrane, and is coupled to the generation of an electrochemical proton potential (Deltap) across the bacterial membrane. The experimental evidence concerning the electron transport and its coupling to Deltap generation is reviewed in this article. The electron transport chain consists of fumarate reductase, menaquinone (MK) and either hydrogenase or formate dehydrogenase. Measurements indicate that the Deltap is generated exclusively by MK reduction with H2 or formate; MKH2 oxidation by fumarate appears to be an electroneutral process. However, evidence derived from the crystal structure of fumarate reductase suggests an electrogenic mechanism for the latter process.

Hydrogenase and fumarate reductase isolated from Woli-nella succinogenes were incorporated into liposomes containing menaquinone. The two enzymes were found to be oriented solely to the outside of the resulting proteolipo-somes. The proteoliposomes catalyzed fumarate reduction by H 2 which generated an electrical proton potential (Dw ¼ 0.19 V, negative inside) in the same direction as that generated by fumarate respiration in cells of W. succinogenes. The H + /e ratio brought about by fumarate reduction with H 2 in proteoliposomes in the presence of valinomycin and external K + was approximately 1. The same Dw and H + /e ratio was associated with the reduction of 2,3-dimethyl-1,4-naphthoquinone (DMN) by H 2 in proteoliposomes containing menaquinone and hydrogenase with or without fumarate reductase. Proteoliposomes containing menaqui-none and fumarate reductase with or without hydrogenase catalyzed fumarate reduction by DMNH 2 which did not generate a Dw. Incorporation of formate dehydrogenase together with fumarate reductase and menaquinone resulted in proteoliposomes catalyzing the reduction of fumarate or DMN by formate. Both reactions generated a Dw of 0.13 V (negative inside). The H + /e ratio of formate oxidation by menaquinone or DMN was close to 1. The results demonstrate for the first time that coupled fumarate respiration can be restored in liposomes using the well characterized electron transport enzymes isolated from W. succinogenes. The results support the view that Dw generation is coupled to menaquinone reduction by H 2 or formate, but not to menaquinol oxidation by fumarate. Dw generation is probably caused by proton uptake from the cytoplasmic side of the membrane during menaquinone reduction, and by the coupled release of protons from H 2 or formate oxidation on the periplasmic side. This mechanism is supported by the properties of two hydrogenase mutants of W. succinogenes which indicate that the site of quinone reduction is close to the cytoplasmic surface of the membrane.

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Modi¢cation of heme c binding motifs in the small subunit (NrfH) of the Wolinella succinogenes cytochrome c nitrite reductase complex IntroductionWolinella succinogenes can grow by respiratory nitrite ammoni¢cation using formate as electron donor [1,2]. The electron transport chain catalyzing this reaction consists of two membrane-bound enzyme complexes and menaquinone. The formate dehydrogenase complex catalyzes the reduction of menaquinone by formate, and menaquinol oxidation by nitrite is catalyzed by the cytochrome c nitrite reductase complex. The former reaction is known to be coupled to the generation of an electrochemical proton potential [3^5], whereas menaquinol oxidation by nitrite appears to be an electroneutral process [2,6,7]. The cytochrome c nitrite reductase complex consists of two c-type cytochromes, the pentaheme catalytic subunit NrfA and the tetraheme subunit NrfH [6^9]. NrfH anchors the complex in the bacterial membrane and mediates electron transport from menaquinol to NrfA [6,7].NrfH and NrfA are encoded by the ¢rst two genes of the nrfHAIJ operon [6]. The nrfI gene product was shown to be required for the attachment of the active site heme group to NrfA [10] while the function of the nrfJ gene is unknown. A vnrfJ deletion mutant of W. succinogenes had wild-type properties with respect to nitrite respiration [6]. Recently, a genetic system has been described that allows expression of mutated nrfH alleles in W. succinogenes [7]. The procedure is based on the integration of a nrfH-containing plasmid into the genome of the W. succinogenes vnrfH deletion mutant thus restoring the nrfHAIJ operon.NrfH belongs to the NapC/NirT family of multiheme c-type cytochromes [2,7,11]. Members of this protein family are commonly found in bacteria and are generally thought to mediate electron transport from a respiratory quinone to a periplasmic oxidoreductase. To date, no high-resolution structure of any member of the NapC/NirT family is available although crystals of the W. succinogenes NrfHA complex have been obtained recently [12]. Furthermore, no data on site-directed modi¢cation of any NapC/NirT-like cytochrome have yet been reported. NrfH contains four heme c binding motifs (CXXCH) to which heme is covalently attached by forming two thioether bonds [7]. The detailed function of the four heme groups is unclear. It is not known how they are arranged in the protein or whether all four hemes are involved in electron transfer from menaquinol to NrfA. In this communication, the role of the NrfH heme groups was investigated by replacing the cysteine residues of the heme c binding motifs. Materials and methods2.1. Growth of W. succinogenes, cell fractionation and puri¢cation of the nitrite reductase complex Strains of W. succinogenes were grown with formate and nitrate as described [13] but (NH 4 ) 2 SO 4 (5 mM) was present in the medium instead of K 2 SO 4 . The medium was supplemented with brain-heartinfusion broth (0.5%, w/v) which was only left out when the capability of growth by nitrite respiration wa...

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