Respiration in cyanobacterial thylakoid membranes is interwoven with photosynthetic processes. We have constructed a range of mutants that are impaired in several combinations of respiratory and photosynthetic electron transport complexes and have examined the relative effects on the redox state of the plastoquinone (PQ) pool by using a quinone electrode. Succinate dehydrogenase has a major effect on the PQ redox poise, as mutants lacking this enzyme showed a much more oxidized PQ pool. Mutants lacking type I and II NAD(P)H dehydrogenases also had more oxidized PQ pools. However, in the mutant lacking type I NADPH dehydrogenase, succinate was essentially absent and effective respiratory electron donation to the PQ pool could be established after addition of 1 mM succinate. Therefore, lack of the type I NADPH dehydrogenase had an indirect effect on the PQ pool redox state. The electron donation capacity of succinate dehydrogenase was found to be an order of magnitude larger than that of type I and II NAD(P)H dehydrogenases. The reason for the oxidized PQ pool upon inactivation of type II NADH dehydrogenase may be related to the facts that the NAD pool in the cell is much smaller than that of NADP and that the NAD pool is fully reduced in the mutant without type II NADH dehydrogenase, thus causing regulatory inhibition. The results indicate that succinate dehydrogenase is the main respiratory electron transfer pathway into the PQ pool and that type I and II NAD(P)H dehydrogenases regulate the reduction level of NADP and NAD, which, in turn, affects respiratory electron flow through succinate dehydrogenase.

Multiple instances of low potential electron transport pathway inhibitors that affect the structure of the cytochrome (cyt) bc 1 complex to varying degrees, ranging from changes in hydroquinone (QH 2 ) oxidation and cyt c 1 reduction kinetics, to proteolytic accessibility of the hinge region of the iron-sulfur containing subunit (Fe/S protein), have been reported. However, no instance has been documented of any ensuing change on the environment(s) of the [2Fe-2S] cluster. In this work, this issue was addressed in detail by taking advantage of the increased spectral and spatial resolution obtainable with orientation dependent electron paramagnetic resonance (EPR) spectroscopic analysis of ordered membrane preparations. For the first time, perturbation of the low potential electron transport pathway by Q i site inhibitors or various mutations was shown to change the EPR spectra of both the cyt b hemes and the [2Fe-2S] cluster of the Fe/S protein. In particular, two interlinked effects of Q i site modifications on the Fe/S subunit, one changing the local environment of its [2Fe-2S] cluster, and a second affecting the mobility of this subunit are revealed. Remarkably, different inhibitors and mutations at or near the Q i site induce these two effects differently, indicating that the events occurring at the Q i site affect the global structure of the cyt bc 1. Furthermore, occupancy of discrete Q i site subdomains differently impede the location of the Fe/S protein at the Q o site. These findings led us to propose that antimycin A and HQNO mimic the presence of QH 2 and Q at the Q i site, respectively. Implications of these findings in respect to the Q o -Q i sites communications and to multiple turnovers of the cyt bc 1 are discussed. KeywordsAntimycin A; cytochrome bc 1 ; complex III; Rhodobacter capsulatus; photosynthesis and respiration; energy transduction ABBREVIATIONSElectron paramagnetic resonance (EPR); [2Fe-2S] cluster containing protein (Fe/S); quinone (Q); cytochrome b (cyt b); hydroquinone (QH 2 ); hydroquinone:cytochrome (cyt) c oxidoreductase (cyt bc 1 ); 2-heptyl-4-hydroxyquinoline N-oxide (HQNO); 2-nonyl-4-hydroxyquinoline N-oxide (NQNO); quinone reduction site (Q i ); hydroquinone oxidation site (Q o ) *To whom correspondence should be addressed: Phone: (215) 898-4394 Fax: (215) 898-8780 E-mail:fdaldal@sas.upenn.edu. This work was supported by NIH grant R01 GM 38237 to F. D. and NIH F32 GM 65791 and AHA #0425515U fellowships to J. W. C. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2006 February 2. Published in final edited form as:Biochemistry. 2005 August 9; 44(31): 10520-10532. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptThe hydroquinone (QH 2 ):cytochrome (cyt) c oxidoreductase (cyt bc 1 ) is an essential component of the mitochondrial and most bacterial respiratory electron transport pathways (1). A sister complex, the cyt b 6 f, is also a part of the photosynthetic electron transport chains of the chloroplasts of highe...

The open reading frames sll1625 and sll0823, which have significant sequence similarity to genes coding for the FeS subunits of succinate dehydrogenase and fumarate reductase, were deleted singly and in combination in the cyanobacterium Synechocystis sp. strain PCC 6803. When the organic acid content in the ⌬sll1625 and ⌬sll0823 strains was analyzed, a 100-fold decrease in succinate and fumarate concentrations was observed relative to the wild type. A similar analysis for the ⌬sll1625 ⌬sll0823 strain revealed that 17% of the wild-type succinate levels remained, while only 1 to 2% of the wild-type fumarate levels were present. Addition of 2-oxoglutarate to the growth media of the double mutant strain prior to analysis of organic acids in cells caused succinate to accumulate. This indicates that succinate dehydrogenase activity had been blocked by the deletions and that 2-oxoglutarate can be converted to succinate in vivo in this organism, even though a traditional 2-oxoglutarate dehydrogenase is lacking. In addition, reduction of the thylakoid plastoquinone pool in darkness in the presence of KCN was up to fivefold slower in the mutants than in the wild type. Moreover, in vitro succinate dehydrogenase activity observed in wild-type membranes is absent from those isolated from the double mutant and reduced in those from the single mutants, further indicating that the sll1625 and sll0823 open reading frames encode subunits of succinate dehydrogenase complexes that are active in the thylakoid membrane of the cyanobacterium.

Atovaquone is an antiparasitic drug that selectively inhibits electron transport through the parasite mitochondrial cytochrome bc 1 complex and collapses the mitochondrial membrane potential at concentrations far lower than those at which the mammalian system is affected. Because this molecule represents a new class of antimicrobial agents, we seek a deeper understanding of its mode of action. To that end, we employed site-directed mutagenesis of a bacterial cytochrome b, combined with biophysical and biochemical measurements. A large scale domain movement involving the iron-sulfur protein subunit is required for electron transfer from cytochrome b-bound ubihydroquinone to cytochrome c 1 of the cytochrome bc 1 complex. Here, we show that atovaquone blocks this domain movement by locking the iron-sulfur subunit in its cytochrome b-binding conformation. Based on our malaria atovaquone resistance data, a series of cytochrome b mutants was produced that were predicted to have either enhanced or reduced sensitivity to atovaquone. Mutations altering the bacterial cytochrome b at its ef loop to more closely resemble Plasmodium cytochrome b increased the sensitivity of the cytochrome bc 1 complex to atovaquone. A mutation within the ef loop that is associated with resistant malaria parasites rendered the complex resistant to atovaquone, thereby providing direct proof that the mutation causes atovaquone resistance. This mutation resulted in a 10-fold reduction in the in vitro activity of the cytochrome bc 1 complex, suggesting that it may exert a cost on efficiency of the cytochrome bc 1 complex.Malaria is one of the most intractable human afflictions in the world. Despite intensive campaigns against the parasitic disease, an estimated 300 to 500 million cases still occur each year. Plasmodium falciparum, the causative agent of the most lethal form of malaria, is responsible for over 2 million deaths annually, largely among young children and pregnant women (1, 2). Efforts to eradicate malaria have not been successful, and the situation may be worsening due, in large part, to the emergence and spread of drug-resistant parasites. The need for new antimalarial drugs is now widely recognized. Atovaquone represents a new class of drugs having a metabolic target different from extant antimalarial drugs to which resistance is widespread. Atovaquone was found to be a very effective antimalarial compound, but unsuitable for use as a single agent because of the relatively quick emergence of resistance (3-5). However, in combination with the synergistic agent proguanil, it has been effective for both therapeutic and prophylactic uses (5-7). On the other hand, once atovaquone resistance has arisen, the combination is no longer effective against malaria parasites (8,9).Previous studies have shown that atovaquone selectively inhibits mitochondrial electron transport in the parasite, consistent with the prevalent theory that hydroxynaphthoquinones function as ubiquinone antagonists (10, 11). Additionally, atovaquone was found to colla...