This study shows that the product of the hoxZ gene of Alcaligenes eutrophus HI6 is a 6-type cytochrome (cytochrome bJ, which is essential for anchoring the membrane-bound hydrogenase (MBH) complex to the periplasmic side of the membrane and for H,-coupled respiration. The hoxZ product is not required for MBH translocation and H,-dependent reduction of the redox dye, 2,3,5-triphenyl-2-tetrazolium chloride. The lack of cytochrome b, does not affect the electron-transport activities linked to oxidation of succinate and NADH, although it enhances the electron-flow rate through the cytochrome-c oxidase pathway in hoxZA membranes. We show that the hoxZ product is a dihaem cytochrome b (haems with of + 10 mV and + 166 mV) involved in H,-dependent electron transfer. We conclude that cytochrome b, of the A. eutrophus MBH complex is the link necessary for transfer of electrons from H, to the ubiquinone pool and that it is required for attachment of MBH to the membrane.Keywords: cytochrome b subunit; hydrogen respiration ; hoxZ gene; membrane-bound hydrogenase; Alcaligenes eutrophus.Alcaligenes eutrophus H16, a gram-negative respiration-dependent bacterium, belongs to the group of facultative lithoautotrophs that can use hydrogen as sole energy source [l]. Oxidation of hydrogen is mediated by two [NiFeI-containing hydrogenases : a cytoplasmic, heterotetrameric NAD-reducing enzyme (SH), which consists of four subunits encoded by the genes hoxF, hoxU, hoxH and hoxY [ 2 ] ; and a membrane-bound hydrogenase (MBH), which is composed of a small and a large subunit, encoded by the genes hoxK and hoxC, respectively [3]. The structural genes for SH and MBH are arranged in two separate operons tightly clustered with sets of accessory genes that are required for the formation of enzymatically active hydrogenase. The accessory-gene products participate in a series of complex post-translational events involving metal-center assembly, C-terminal proteolytic processing, oligomerization and, in the case of the MBH, translocation [4 -61.Crystal-structure analysis of the periplasmic [NiFe] hydrogenase of Desulfovibrio gigas has shown that the binuclear metal center is deep inside the protein and that the mature large subunit is devoid of a C-terminal extension [7]. Evidence for the presence of specific proteases that remove at least 15 C-terminal residues has been documented in a variety of phylogenetically distant species, including A. eutrophus [X- nological assays revealed that cells of A. eutrophus, containing active MBH, produce the small and large subunits of the enzyme in two electrophoretically distinct conformations. It was suggested that the conversion of the two subunits into the catalytically active membrane-associated heterodimer is dependent on the function of accessory-gene products [12]. More recently, we described two classes of mutants with in-frame deletions in the eight MBH-linked accessory genes. Class-I mutants, affected in hoxM, hoxO and hoxQ, are totally devoid of MBH activity, whereas class-I1 mutants, harboring del...

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We report the first detailed study on the ubiquinone (coenzyme Q; abbreviated to Q) analogue specificity of mitochondrial complex I, NADH:Q reductase, in intact submitochondrial particles. The enzymic function of complex I has been investigated using a series of analogues of Q as electron acceptor substrates for both electron transport activity and the associated generation of membrane potential. Q analogues with a saturated substituent of one to three carbons at position 6 of the 2,3-dimethoxy-5-methyl-1,4-benzoquinone ring have the fastest rates of electron transport activity, and analogues with a substituent of seven to nine carbon atoms have the highest values of association constant derived from NADH:Q reductase activity. The rate of NADH:Q reductase activity is potently but incompletely inhibited by rotenone, and the residual rotenone-insensitive rate is stimulated by Q analogues in different ways depending on the hydrophobicity of their substituent. Membrane potential measurements have been undertaken to evaluate the energetic efficiency of complex I with various Q analogues. Only hydrophobic analogues such as nonyl-Q or undecyl-Q show an efficiency of membrane potential generation equivalent to that of endogenous Q. The less hydrophobic analogues as well as the isoprenoid analogue Q-2 are more efficient as substrates for the redox activity of complex I than for membrane potential generation. Thus the hydrophilic Q analogues act also as electron sinks and interact incompletely with the physiological Q site in complex I that pumps protons and generates membrane potential.

Plasma membranes isolated from cells of the halophilic purple nonsulfur bacterium Rhodospirillum salinarum grown in light or in the dark were examined. Membranes isolated from cells grown aerobically in the dark contained three b-type and two c-type membrane-bound cytochromes with Em,7 of +180, +72 and -5 mV (561-575 nm), and +244 and +27 mV (551-540 nm), respectively. Conversely, membranes isolated from cells grown anaerobically in the light contained two b-type and five c-type haems with Em,7 of +60 and -45 mV and +290, +250, +135, -20 and -105 mV, respectively. In addition to haems of the b- and c-type, two haems of the a-type (Em,7 of +325 and +175 mV) were present only in cells grown in the dark. Four soluble cytochromes of the c type, but not cytochrome c2, along with two high-potential iron-sulfur proteins (HiPIP iso-1 and iso-2) were also identified in cells grown aerobically. Inhibitory studies showed that 85-90% of the respiratory activity was blocked by very low concentrations of cyanide, antimycin A and myxothiazol (50, 0.1 and 0.2 mM, respectively). These results taken together were interpreted to show that the oxidative electron transport chain of Rsp. salinarum is linear, leading to a membrane-bound oxidase of the aa3 type in cells grown in the dark, while no significant cytochrome oxidase activity is catalyzed by photosynthetic membranes. These features suggest that this halophilic species is unique among the genus Rhodospirillum and that it also differs from other facultative phototrophs (e.g., Rhodobacter species) in that it does not contain either cytochrome c2 or a branched respiratory chain.

To investigate the energy-conserving function of the NADH:ubiquinone reductase (complex I), we have selected oxonol VI [bis(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol] as the most sensitive probe for measuring the reactions of membrane potential generation in submitochondrial particles. Calibration of the oxonol signals with potassium diffusion potentials shows a non-linear response after a threshold around -50 mV. Thermodynamic evaluations indicate that the upper limit of the oxonol response to the potential generated by complex I is around -220 mV, which is close to the maximal protonmotive force in coupled submitochondrial particles. NADH addition to particles in which ubiquinol oxidation is blocked by inhibitors of other respiratory complexes generates oxonol signals corresponding to membrane potentials of -130 to -180 mV. These signals are produced by about four turnovers of the complex reducing endogenous ubiquinone (i.e. non-steady-state conditions) and are equivalent to a charge separation similar to that of the antimycin-sensitive reactions of ubiquinol:cytochrome c reductase (complex III). The transient oxonol signals under non-steady-state conditions are thus informative of crucial steps in the electrogenic reactions catalyzed by complex I. The possible nature of these electrogenic reactions is discussed in relation to proposed mechanisms for complex I.