Inhibition of colicin synthesis by the antibiotic globomycin

Wolinella succinogenes can grow by anaerobic respiration with fumarate or polysulfide as the terminal electron acceptor, and H₂ or formate as the electron donor. A ΔhydABC mutant lacking the hydrogenase structural genes did not grow with H₂ and either fumarate or polysulfide. In contrast to the wild-type strain, the mutant grown with fumarate and with formate instead of H₂ did not catalyze the reduction of fumarate, polysulfide, dimethylnaphthoquinone, or benzyl viologen by H₂. Growth and enzymic activities were restored upon integration of a plasmid carrying hydABC into the genome of the ΔhydABC mutant. The ΔhydABC mutant was complemented with hydABC operons modified by artificial stop codons in hydA (StopA) or at the 5′-end of hydC (StopC). The StopC mutant lacked HydC, and the hydrophobic C-terminus of HydA was missing in the hydrogenase of the StopA mutant. The two mutants catalyzed benzyl viologen reduction by H₂. The enzyme activity was located in the membrane of the mutants. A mutant with both modifications (StopAC) contained the activity in the periplasm. The three mutants did not grow with H₂ and either fumarate or polysulfide, and did not catalyze dimethylnaphthoquinone reduction by H₂. We conclude that the same hydrogenase serves in the anaerobic respiration with fumarate and with polysulfide. HydC and the C-terminus of HydA appear to be required for both routes of electron transport and for dimethylnaphthoquinone reduction by H₂. The hydrogenase is anchored in the membrane by HydC and by the C-terminus of HydA. The catalytic subunit HydB is oriented towards the periplasmic side of the membrane. Received: 29 December 1997 / Accepted: 6 March 1998     

The role of the twin-arginine motif in the signal peptide encoded by the hydA gene of the hydrogenase from Wolinella succinogenes

The hydABC operon of Wolinella succinogenes encodes the three subunits of the membrane-integrated Ni-hydrogenase. The catalytic subunit, HydB, is on the periplasmic side of the membrane. Residues R41 and R42 of the twin-arginine motif within the signal peptide of the precursor of the iron-sulfur subunit, HydA, were replaced by two glutamine residues. The corresponding mutant did not grow with H₂ as the electron donor of anaerobic respiration. Mature HydB and the precursor protein of HydA were located exclusively in the cytoplasmic cell fraction of the mutant, which catalyzed the reduction of benzyl viologen by H₂, suggesting that HydB contained Ni. The HydC protein was located in the membrane fraction of the mutant in wild-type amounts. HydC was purified and was shown to contain heme. The results suggest that HydA and HydB are translocated across the membrane by the Tat (twin-arginine translocation) system. The translocation of HydA and HydB as well as the maturation of the precursor protein of HydA appear to depend on the presence of the twin-arginine motif. In contrast, maturation of HydB, the insertion of HydC into the membrane, and heme attachment to HydC are apparently independent of the twin-arginine motif and do not require translocation of the two other hydrogenase subunits. Received: 17 June 1999 / Accepted: 21 July 1999     

Role of His residues in Bacillus subtilis cytochrome b558 for haem binding and assembly of succinate: quinone oxidoreductase (complex II)

Cytochrome b558 in the cytoplasmic membrane of Bacillus subtilis constitutes the anchor and electron acceptor to the flavoprotein (Fp) and iron-sulphur protein (Ip) in succinate:quinone oxidoreductase, and seemingly contains two haem groups. EPR and MCD spectroscopic data indicate bis-imidazole ligation of the haem. Apo-cytochrome was found in the membrane fraction of haem-deficient B. subtilis, suggesting that during biogenesis of the oxidoreductase the cytochrome b558 polypeptide is embedded into the membrane prior to the incorporation of haem and subsequent binding of Fp and Ip. The six His residues in cytochrome b558 were individually changed to Tyr to attempt identification of residues serving as haem axial ligands and to analyse the role of His residues for assembly and function of the oxidoreductase. From the properties of the mutants, His-47 can be excluded as a haem ligand. The remaining His residues (at positions 13, 28, 70, 113 and 155) are located in or close to four predicted transmembrane segments. The Tyr-28 and Tyr-70 mutant proteins appeared to lack one of the two haems. Only the Tyr-13 and Tyr-47 mutant cytochromes were found to function as anchors for Fp and Ip, but the Tyr-13 mutant cytochrome assembles into an enzymatically defective succinate:quinone oxidoreductase. It is concluded from a combination of the experimental findings, sequence comparisons and membrane topology data that His-28, His-70 and His-155 are probably haem axial ligands in a dihaem cytochrome b558. His-70 and His-155 may be ligands to the same haem.     

Hydrogenase Activity and the H2-Fumarate Electron Transport System in Bacteroides fragilis

Hydrogenase activity and the H₂-fumarate electron transport system in a carbohydrate-fermenting obligate anaerobe, Bacteroides fragilis, were investigated. In both whole cells and cell extracts, hydrogenase activity was demonstrated with methylene blue, benzyl viologen, flavin mononucleotide, or flavin adenine dinucleotide as the electron acceptor. A catalytic quantity of benzyl viologen or ferredoxin from Clostridium pasteurianum was required to reduce nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate with H₂. Much of the hydrogenase activity appeared to be associated with the soluble fraction of the cell. Fumarate reduction to succinate by H₂ was demonstrable in cell extracts only in the presence of a catalytic quantity of benzyl viologen, flavin mononucleotide, flavin adenine dinucleotide, or ferredoxin from C. pasteurianum. Sulfhydryl compounds were not required for fumarate reduction by H₂, but mercaptoethanol and dithiothreitol appeared to stimulate this activity by 59 and 61%, respectively. Inhibition of fumarate reduction by acriflavin, rotenone, 2-heptyl-4-hydroxyquinoline-N-oxide, and antimycin A suggest the involvement of a flavoprotein, a quinone, and cytochrome b in the reduction of fumarate to succinate. The involvement of a quinone in fumarate reduction is also apparent from the inhibition of fumarate reduction by H₂ when cell extracts were irradiated with ultraviolet light. Based on the evidence obtained, a possible scheme for the flow of electrons from H₂ to fumarate in B. fragilis is proposed.     

Unification of [FeFe]-hydrogenases into three structural and functional groups

Background[FeFe]-hydrogenases (Hyd) are structurally diverse enzymes that catalyze the reversible oxidation of hydrogen (H₂). Recent biochemical data demonstrate new functional roles for these enzymes, including those that function in electron bifurcation where an exergonic reaction is coupled with an endergonic reaction to drive the reversible oxidation/production of H₂.MethodsTo identify the structural determinants that underpin differences in enzyme functionality, a total of 714 homologous sequences of the catalytic subunit, HydA, were compiled. Bioinformatics approaches informed by biochemical data were then used to characterize differences in inferred quaternary structure, HydA active site protein environment, accessory iron-sulfur clusters in HydA, and regulatory proteins encoded in HydA gene neighborhoods.ResultsHydA homologs were clustered into one of three classification groups, Group 1 (G1), Group 2 (G2), and Group 3 (G3). G1 enzymes were predicted to be monomeric while those in G2 and G3 were predicted to be multimeric and include HydB, HydC (G2/G3) and HydD (G3) subunits. Variation in the HydA active site and accessory iron-sulfur clusters did not vary by group type. Group-specific regulatory genes were identified in the gene neighborhoods of both G2 and G3 Hyd. Analyses of purified G2 and G3 enzymes by mass spectrometry strongly suggest that they are post-translationally modified by phosphorylation.ConclusionsThese results suggest that bifurcation capability is dictated primarily by the presence of both HydB and HydC in Hyd complexes, rather than by variation in HydA.General significanceThis classification scheme provides a framework for future biochemical and mutagenesis studies to elucidate the functional role of Hyd enzymes.     

Physiology and Bioenergetics of [NiFe]-Hydrogenase 2-Catalyzed H2-Consuming and H2-Producing Reactions in Escherichia coli

Escherichia coli uptake hydrogenase 2 (Hyd-2) catalyzes the reversible oxidation of H₂ to protons and electrons. Hyd-2 synthesis is strongly upregulated during growth on glycerol or on glycerol-fumarate. Membrane-associated Hyd-2 is an unusual heterotetrameric [NiFe]-hydrogenase that lacks a typical cytochrome b membrane anchor subunit, which transfers electrons to the quinone pool. Instead, Hyd-2 has an additional electron transfer subunit, termed HybA, with four predicted iron-sulfur clusters. Here, we examined the physiological role of the HybA subunit. During respiratory growth with glycerol and fumarate, Hyd-2 used menaquinone/demethylmenaquinone (MQ/DMQ) to couple hydrogen oxidation to fumarate reduction. HybA was essential for electron transfer from Hyd-2 to MQ/DMQ. H₂ evolution catalyzed by Hyd-2 during fermentation of glycerol in the presence of Casamino Acids or in a fumarate reductase-negative strain growing with glycerol-fumarate was also shown to be dependent on both HybA and MQ/DMQ. The uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) inhibited Hyd-2-dependent H₂ evolution from glycerol, indicating the requirement for a proton gradient. In contrast, CCCP failed to inhibit H₂-coupled fumarate reduction. Although a Hyd-2 enzyme lacking HybA could not catalyze Hyd-2-dependent H₂ oxidation or H₂ evolution in whole cells, reversible H₂-dependent reduction of viologen dyes still occurred. Finally, hydrogen-dependent dye reduction by Hyd-2 was reversibly inhibited in extracts derived from cells grown in H₂ evolution mode. Our findings suggest that Hyd-2 switches between H₂-consuming and H₂-producing modes in response to the redox status of the quinone pool. Hyd-2-dependent H₂ evolution from glycerol requires reverse electron transport.     

Mutations in cytochrome b that affect kinetics of the electron transfer reactions at center N in the yeast cytochrome bc1 complex

We have examined the pre-steady-state kinetics and thermodynamic properties of the b hemes in variants of the yeast cytochrome bc₁ complex that have mutations in the quinone reductase site (center N). Trp-30 is a highly conserved residue, forming a hydrogen bond with the propionate on the high potential b heme (b_H heme). The substitution by a cysteine (W30C) lowers the redox potential of the heme and an apparent consequence is a lower rate of electron transfer between quinol and heme at center N. Leu-198 is also in close proximity to the b_H heme and a L198F mutation alters the spectral properties of the heme but has only minor effects on its redox properties or the electron transfer kinetics at center N. Substitution of Met-221 by glutamine or glutamate results in the loss of a hydrophobic interaction that stabilizes the quinone ligands. Ser-20 and Gln-22 form a hydrogen-bonding network that includes His-202, one of the carbonyl groups of the ubiquinone ring, and an active-site water. A S20T mutation has long-range structural effects on center P and thermodynamic effects on both b hemes. The other mutations (M221E, M221Q, Q22E and Q22T) do not affect the ubiquinol oxidation kinetics at center P, but do modify the electron transfer reactions at center N to various extents. The pre-steady reduction kinetics suggest that these mutations alter the binding of quinone ligands at center N, possibly by widening the binding pocket and thus increasing the distance between the substrate and the b_H heme. These results show that one can distinguish between the contribution of structural and thermodynamic factors to center N function.     

Electron transport driven phosphorylation catalyzed by proteoliposomes containing hydrogenase,fumarate reductase and ATP synthase

Liposomes containing the hydrogenase complex and the fumarate reductase complex isolated from Wolinella (formerly Vibrio) succinogenes, together with vitamin K₁ catalyzed the electron transport from H₂ to fumarate. With the fumarate reductase complex present in excess the activity of electron transport was close to that of the hydrogenase complex. Liposomes containing an ATP synthase in addition to the electron transport components catalyzed the phosphorylation of ADP driven by the electron transport.     

Wolinella succinogenes fumarate reductase contains a dihaem cytochrome b

The fumarate reductase operon of Wolinella succinogenes is made up of three structural genes (frd-CAB). The frdC gene was located next to the promoter region and identified as the cytochrome b structural gene encoding 256 amino acid residues. The N-terminal amino acid sequences of seven fragments derived from the cytochrome b moiety of the enzyme all mapped within the frdC gene. This suggested that the enzyme contained only one species of cytochrome b. Re-evaluation of earlier measurements of subunit composition, haem B content and molecular weight led to the conclusion that the enzyme contained one molecule of cytochrome b with two haem B groups. The hydropathy plot of the amino acid sequence predicted five membrane-spanning hydrophobic segments, the first four of which contained a single histidine residue each. These residues could form the axial ligands to the two haem B groups. FrdC was found to be homologous with the cytochrome b (SdhC) of the Bacillus subtilis succinate dehydrogenase, but not with the hydrophobic subunits of the fumarate reductase or succinate dehydrogenase of Escherichia coli.     

Characterization of the Key Step for Light-driven Hydrogen Evolution in Green Algae

Under anaerobic conditions, several species of green algae perform a light-dependent hydrogen production catalyzed by a special group of [FeFe] hydrogenases termed HydA. Although highly interesting for biotechnological applications, the direct connection between photosynthetic electron transport and hydrogenase activity is still a matter of speculation. By establishing an in vitro reconstitution system, we demonstrate that the photosynthetic ferredoxin (PetF) is essential for efficient electron transfer between photosystem I and HydA1. To investigate the electrostatic interaction process and electron transfer between PetF and HydA1, we performed site-directed mutagenesis. Kinetic analyses with several site-directed mutagenesis variants of HydA1 and PetF enabled us to localize the respective contact sites. These experiments in combination with in silico docking analyses indicate that electrostatic interactions between the conserved HydA1 residue Lys³⁹⁶ and the C terminus of PetF as well as between the PetF residue Glu¹²² and the N-terminal amino group of HydA1 play a major role in complex formation and electron transfer. Mapping of relevant HydA1 and PetF residues constitutes an important basis for manipulating the physiological photosynthetic electron flow in favor of light-driven H₂ production.     

Investigation of the fumarate metabolism of the syntrophic propionate-oxidizing bacterium strain MPOB

The growth of the syntrophic propionate-oxidizing bacterium strain MPOB in pure culture by fumarate disproportionation into carbon dioxide and succinate and by fumarate reduction with propionate, formate or hydrogen as electron donor was studied. The highest growth yield, 12.2 g dry cells/mol fumarate, was observed for growth by fumarate disproportionation. In the presence of hydrogen, formate or propionate, the growth yield was more than twice as low: 4.8, 4.6, and 5.2 g dry cells/mol fumarate, respectively. The location of enzymes that are involved in the electron transport chain during fumarate reduction in strain MPOB was analyzed. Fumarate reductase, succinate dehydrogenase, and ATPase were membrane-bound, while formate dehydrogenase and hydrogenase were loosely attached to the periplasmic side of the membrane. The cells contained cytochrome c, cytochrome b, menaquinone-6 and menaquinone-7 as possible electron carriers. Fumarate reduction with hydrogen in membranes of strain MPOB was inhibited by 2-(heptyl)-4-hydroxyquinoline-N-oxide (HOQNO). This inhibition, together with the activity of fumarate reductase with reduced 2,3-dimethyl-1,4-naphtoquinone (DMNH₂) and the observation that cytochrome b of strain MPOB was oxidized by fumarate, suggested that menequinone and cytochrome b are involved in the electron transport during fumarate reduction in strain MPOB. The growth yields of fumarate reduction with hydrogen or formate as electron donor were similar to the growth yield of Wolinella succinogenes. Therefore, it can be assumed that strain MPOB gains the same amount of ATP from fumarate reduction as W. succinogenes, i.e. 0.7 mol ATP/mol fumarate. This value supports the hypothesis that syntrophic propionate-oxidizing bacteria have to invest two-thirds of an ATP via reversed electron transport in the succinate oxidation step during the oxidation of propionate. The same electron transport chain that is involved in fumarate reduction may operate in the reversed direction to drive the energetically unfavourable oxidation of succinate during syntrophic propionate oxidation since (1) cytochrome b was reduced by succinate and (2) succinate oxidation was similarly inhibited by HOQNO as fumarate reduction. Received: 18 March 1997 / Accepted: 10 November 1997     

Electron sweep across four b-hemes of cytochrome bc1 revealed by unusual paramagnetic properties of the Qi semiquinone intermediate

Dimeric cytochromes bc are central components of photosynthetic and respiratory electron transport chains. In their catalytic core, four hemes b connect four quinone (Q) binding sites. Two of these sites, Q_i sites, reduce quinone to quinol (QH₂) in a step-wise reaction, involving a stable semiquinone intermediate (SQ_i). However, the interaction of the SQ_i with the adjacent hemes remains largely unexplored. Here, by revealing the existence of two populations of SQ_i differing in paramagnetic relaxation, we present a new mechanistic insight into this interaction. Benefiting from a clear separation of these SQ_i species in mutants with a changed redox midpoint potential of hemes b, we identified that the fast-relaxing SQ_i (SQ_iF) corresponds to the form magnetically coupled with the oxidized heme b_H (the heme b adjacent to the Q_i site), while the slow-relaxing SQ_i (SQ_iS) reflects the form present alongside the reduced (and diamagnetic) heme b_H. This so far unreported SQ_iF calls for a reinvestigation of the thermodynamic properties of SQ_i and the Q_i site. The existence of SQ_iF in the native enzyme reveals a possibility of an extended electron equilibration within the dimer, involving all four hemes b and both Q_i sites. This substantiates the predicted earlier electron transfer acting to sweep the b-chain of reduced hemes b to diminish generation of reactive oxygen species by cytochrome bc₁. In analogy to the Q_i site, we anticipate that the quinone binding sites in other enzymes may contain yet undetected semiquinones which interact magnetically with oxidized hemes upon progress of catalytic reactions.     

Hydrogen Metabolism in Shewanella oneidensis MR-1

Shewanella oneidensis MR-1 is a facultative sediment microorganism which uses diverse compounds, such as oxygen and fumarate, as well as insoluble Fe(III) and Mn(IV) as electron acceptors. The electron donor spectrum is more limited and includes metabolic end products of primary fermenting bacteria, such as lactate, formate, and hydrogen. While the utilization of hydrogen as an electron donor has been described previously, we report here the formation of hydrogen from pyruvate under anaerobic, stationary-phase conditions in the absence of an external electron acceptor. Genes for the two S. oneidensis MR-1 hydrogenases, hydA, encoding a periplasmic [Fe-Fe] hydrogenase, and hyaB, encoding a periplasmic [Ni-Fe] hydrogenase, were found to be expressed only under anaerobic conditions during early exponential growth and into stationary-phase growth. Analyses of ΔhydA, ΔhyaB, and ΔhydA ΔhyaB in-frame-deletion mutants indicated that HydA functions primarily as a hydrogen-forming hydrogenase while HyaB has a bifunctional role and represents the dominant hydrogenase activity under the experimental conditions tested. Based on results from physiological and genetic experiments, we propose that hydrogen is formed from pyruvate by multiple parallel pathways, one pathway involving formate as an intermediate, pyruvate-formate lyase, and formate-hydrogen lyase, comprised of HydA hydrogenase and formate dehydrogenase, and a formate-independent pathway involving pyruvate dehydrogenase. A reverse electron transport chain is potentially involved in a formate-hydrogen lyase-independent pathway. While pyruvate does not support a fermentative mode of growth in this microorganism, pyruvate, in the absence of an electron acceptor, increased cell viability in anaerobic, stationary-phase cultures, suggesting a role in the survival of S. oneidensis MR-1 under stationary-phase conditions.     

<Emphasis Type="Italic">Shewanella oneidensis</Emphasis>: a new and efficient System for Expression and Maturation of heterologous [Fe-Fe] Hydrogenase from <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

Background  

The eukaryotic green alga, Chlamydomonas reinhardtii, produces H₂ under anaerobic conditions, in a reaction catalysed by a [Fe-Fe] hydrogenase HydA1. For further biochemical and biophysical studies a suitable expression system of this enzyme should be found to overcome its weak expression in the host organism. Two heterologous expression systems used up to now have several advantages. However they are not free from some drawbacks. In this work we use bacterium Shewanella oneidensis as a new and efficient system for expression and maturation of HydA1 from Chlamydomonas reinhardtii.     

The effect of sulfur compounds on H<Subscript>2</Subscript> evolution/consumption reactions,mediated by various hydrogenases,in the purple sulfur bacterium, <Emphasis Type="Italic">Thiocapsa roseopersicina</Emphasis>

The influence of reduced sulfur compounds (including stored S⁰) on H₂ evolution/consumption reactions in the purple sulfur bacterium, Thiocapsa roseopersicina BBS, was studied using mutants containing only one of the three known [NiFe] hydrogenase enzymes: Hox, Hup or Hyn. The observed effects depended on the kind of hydrogenase involved. The mutant harbouring Hox hydrogenase was able to use S₂O₃²⁻, SO₃²⁻, S²⁻ and S⁰ as electron donors for light-dependent H₂ production. Dark H₂ evolution from organic substrates via Hox hydrogenase was inhibited by S⁰. Under light conditions, endogenous H₂ uptake by Hox or Hup hydrogenases was suppressed by S compounds. СО₂-dependent H₂ uptake by Hox hydrogenase in the light required the additional presence of S compounds, unlike the Hup-mediated process. Dark H₂ consumption via Hyn hydrogenase was connected to utilization of S⁰ as an electron acceptor and resulted in the accumulation of H₂S. In wild type BBS, with high levels of stored S⁰, dark H₂ production from organic substrates was significantly lower, but H₂S accumulation significantly higher, than in the mutant GB1121(Hox⁺). There is a possibility that H₂ produced via Hox hydrogenase is consumed by Hyn hydrogenase to reduce S⁰.     

Synthetic biology for improved hydrogen production in Chlamydomonas reinhardtii

Hydrogen is a clean alternative to fossil fuels. It has applications for electricity generation and transportation and is used for the manufacturing of ammonia and steel. However, today, H₂ is almost exclusively produced from coal and natural gas. As such, methods to produce H₂ that do not use fossil fuels need to be developed and adopted. The biological manufacturing of H₂ may be one promising solution as this process is clean and renewable. Hydrogen is produced biologically via enzymes called hydrogenases. There are three classes of hydrogenases namely [FeFe], [NiFe] and [Fe] hydrogenases. The [FeFe] hydrogenase HydA1 from the model unicellular algae Chlamydomonas reinhardtii has been studied extensively and belongs to the A1 subclass of [FeFe] hydrogenases that have the highest turnover frequencies amongst hydrogenases (21,000 ± 12,000 H₂ s⁻¹ for CaHydA from Clostridium acetobutyliticum). Yet to date, limitations in C. reinhardtii H₂ production pathways have hampered commercial scale implementation, in part due to O₂ sensitivity of hydrogenases and competing metabolic pathways, resulting in low H₂ production efficiency. Here, we describe key processes in the biogenesis of HydA1 and H₂ production pathways in C. reinhardtii. We also summarize recent advancements of algal H₂ production using synthetic biology and describe valuable tools such as high-throughput screening (HTS) assays to accelerate the process of engineering algae for commercial biological H₂ production.     

Rational redesign of the ferredoxin-NADP+-oxido-reductase/ferredoxin-interaction for photosynthesis-dependent H2-production

Utilization of electrons from the photosynthetic water splitting reaction for the generation of biofuels, commodities as well as application in biotransformations requires a partial rerouting of the photosynthetic electron transport chain. Due to its rather negative redox potential and its bifurcational function, ferredoxin at the acceptor side of Photosystem 1 is one of the focal points for such an engineering. With hydrogen production as model system, we show here the impact and potential of redox partner design involving ferredoxin (Fd), ferredoxin-oxido-reductase (FNR) and [FeFe]?hydrogenase HydA1 on electron transport in a future cyanobacterial design cell of Synechocystis PCC 6803. X-ray-structure-based rational design and the allocation of specific interaction residues by NMR-analysis led to the construction of Fd- and FNR-mutants, which in appropriate combination enabled an about 18-fold enhanced electron flow from Fd to HydA1 (in competition with equimolar amounts of FNR) in in vitro assays. The negative impact of these mutations on the Fd-FNR electron transport which indirectly facilitates H₂ production (with a contribution of ≤42% by FNR variants and ≤23% by Fd-variants) and the direct positive impact on the Fd-HydA1 electron transport (≤23% by Fd-mutants) provide an excellent basis for the construction of a hydrogen-producing design cell and the study of photosynthetic efficiency-optimization with cyanobacteria.     

MOA-stilbene: A new tool for investigation of the reactions of the chloroplast cytochrome bf complex

MOA-stilbene is known to be a specific inhibitor of the Q_o site of mammalian cytochrome bc ₁ complex. We show that it also binds to the chloroplast cytochrome bf complex. Binding to the reduced enzyme induces a red-shift of the Soret and visible absorption bands of the haems b. Steady state and single turnover experiments with thylakoid membranes show that MOA-stilbene promotes additional oxidant-induced reduction of the b haems and slows their subsequent dark reoxidation. In single turnover experiments, the associated slow phase of the carotenoid bandshift at 518 nm is only partially decreased in apparent extent and rate. These and other effects are similar to those produced by NQNO, a Q_i site effector, and by analogy indicate that MOA-stilbene should also be primarily a Q_i-site effector of the cytochrome bf complex. MOA-stilbene has less effect on other parts of the photosynthetic chain. This confers an important advantage on MOA-stilbene in that its effects on the cytochrome bf complex can be studied by using Photosystem II to activate turnover. Myxothiazol displays effects on the cytochrome bf complex which are similar to, but much weaker than, those of MOA-stilbene.A Q cycle-based model of turnover of the cytochrome bf complex is presented, which can account for several unusual features of kinetic behaviour.Abbreviations DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - duroquinol 2,3,5,6-tetramethyl-p-benzohydroquinone - E_hx Ambient potential at pHx versus SHE - E_mx Midpoint potential at pH x versus SHE - haem b _H the higher potential haem b of cytochrome b, thought to be associated with the quinone reduction site, Q_i, and sometimes termed haem b _n - haem b _L the lower potential haem of cytochrome b, thought to be associated with the quinol oxidation site, Q_o, and sometimes termed haem b _p - HQNO 2-n-heptyl-4-hydroxyquinoline-N-oxide - MOA-stilbene E--methoxyacrylate-stilbene or (E,E)-methyl 3-methoxy-2-(styrylphenyl)propenoate - NQNO 2-n-nonyl-4-hydroxyquinoline-N-oxide - Q_B (site) the (binding site of the) secondary quinone acceptor of Photosystem II - Q_o site the quinol oxidation site and site of proton output of the bc and bf complexes (also termed the Q_z or Q_p site) - Q_i site the quinone reduction site and site of proton input of the bc and bf complexes (also termed the Q_c, Q_r or Q_n site) - SHE Standard Hydrogen Electrode