Crystal structure of heme A synthase from Bacillus subtilis.

Heme A is an essential cofactor for respiratory terminal oxidases and vital for respiration in aerobic organisms. The final step of heme A biosynthesis is formylation of the C-8 methyl group of heme molecule by heme A synthase (HAS). HAS is a heme-containing integral membrane protein, and its structure and reaction mechanisms have remained unknown. Thus, little is known about HAS despite of its importance. Here we report the crystal structure of HAS from Bacillus subtilis at 2.2-Å resolution. The N- and C-terminal halves of HAS consist of four-helix bundles and they align in a pseudo twofold symmetry manner. Each bundle contains a pair of histidine residues and forms a heme-binding domain. The C-half domain binds a cofactor-heme molecule, while the N-half domain is vacant. Many water molecules are found in the transmembrane region and around the substrate-binding site, and some of them interact with the main chain of transmembrane helix. Comparison of these two domain structures enables us to construct a substrate-heme binding state structure. This structure implies that a completely conserved glutamate, Glu57 in B. subtilis, is the catalytic residue for the formylation reaction. These results provide valuable suggestions of the substrate-heme binding mechanism. Our results present significant insight into the heme A biosynthesis.

Heme/heme redox interaction and resolution of individual optical absorption spectra of the hemes in cytochrome bd from Escherichia coli.

Cytochrome bd is a terminal component of the respiratory chain of Escherichia coli catalyzing reduction of molecular oxygen to water. It contains three hemes, b(558), b(595), and d. The detailed spectroelectrochemical redox titration and numerical modeling of the data reveal significant redox interaction between the low-spin heme b(558) and high-spin heme b(595), whereas the interaction between heme d and either hemes b appears to be rather weak. However, the presence of heme d itself decreases much larger interaction between the two hemes b. Fitting the titration data with a model where redox interaction between the hemes is explicitly included makes it possible to extract individual absorption spectra of all hemes. The alpha- and beta-band reduced-minus-oxidized difference spectra agree with the data published earlier ([22] J.G. Koland, M.J. Miller, R.B. Gennis, Potentiometric analysis of the purified cytochrome d terminal oxidase complex from Escherichia coli, Biochemistry 23 (1984) 1051-1056., and [23] R.M. Lorence, J.G. Koland, R.B. Gennis, Coulometric and spectroscopic analysis of the purified cytochrome d complex of Escherichia coli: evidence for the identification of "cytochrome a(1)" as cytochrome b(595), Biochemistry 25 (1986) 2314-2321.). The Soret band spectra show lambda(max)=429.5 nm, lambda(min) approximately 413 nm (heme b(558)), lambda(max)=439 nm, lambda(min) approximately 400+/-1 nm (heme b(595)), and lambda(max)=430 nm, lambda(min)=405 nm (heme d). The spectral contribution of heme d to the complex Soret band is much smaller than those of either hemes b; the Soret/alpha (DeltaA(430):DeltaA(629)) ratio for heme d is 1.6.

Antibiotics LL-Z1272 identified as novel inhibitors discriminating bacterial and mitochondrial quinol oxidases.

To counter antibiotic-resistant bacteria, we screened the Kitasato Institute for Life Sciences Chemical Library with bacterial quinol oxidase, which does not exist in the mitochondrial respiratory chain. We identified five prenylphenols, LL-Z1272beta, gamma, delta, epsilon and zeta, as new inhibitors for the Escherichia coli cytochrome bd. We found that these compounds also inhibited the E. coli bo-type ubiquinol oxidase and trypanosome alternative oxidase, although these three oxidases are structurally unrelated. LL-Z1272beta and epsilon (dechlorinated derivatives) were more active against cytochrome bd while LL-Z1272gamma, delta, and zeta (chlorinated derivatives) were potent inhibitors of cytochrome bo and trypanosome alternative oxidase. Thus prenylphenols are useful for the selective inhibition of quinol oxidases and for understanding the molecular mechanisms of respiratory quinol oxidases as a probe for the quinol oxidation site. Since quinol oxidases are absent from mammalian mitochondria, LL-Z1272beta and delta, which are less toxic to human cells, could be used as lead compounds for development of novel chemotherapeutic agents against pathogenic bacteria and African trypanosomiasis.

Gramicidin S and polymyxins: the revival of cationic cyclic peptide antibiotics.

Gramicidin S and polymyxins are small cationic cyclic peptides and act as potent antibiotics against Gram-negative and Gram-positive bacteria by perturbing integrity of the bacterial membranes. Screening of a natural antibiotics library with bacterial membrane vesicles identified gramicidin S as an inhibitor of cytochrome bd quinol oxidase and an alternative NADH dehydrogenase (NDH-2) and polymyxin B as an inhibitor of NDH-2 and malate: quinone oxidoreductase. Our studies showed that cationic cyclic peptide antibiotics have novel molecular targets in the membrane and interfere ligand binding on the hydrophobic surface of enzymes. Improvement of the toxicity and optimization of the structures and clinical uses are urgently needed for their effective application in combating drug-resistant bacteria.

Electron transfer processes in subunit I mutants of cytochrome bo quinol oxidase in Escherichia coli.

Cytochrome bo is a terminal quinol oxidase in the aerobic respiratory chain of Escherichia coli. Subunit I binds all four redox centers, and electrons are transferred from quinols to high-spin heme o and Cu(B) through a bound uniquinone-8 and low-spin heme b. To explore the role of conserved charged amino acid residues, we examined the one-electron transfer processes in subunit I mutants. We found that all the mutants examined increased the electron transfer rate from the bound quinone to heme b more than 40-fold. Tyr288 and Lys362 are key residues in the K-channel for charge compensation of the heme o-Cu(B) binuclear center with protons. The Tyr288Phe and Lys362Gln mutants showed 100-fold decreases in heme b-to-heme o electron transfer, accompanied by large increases in the redox potential of heme o. Our results indicate that electromagnetic coupling of hemes is important for facilitated heme-heme electron transfer in cytochrome bo.

Glutamate 107 in subunit I of cytochrome bd from Escherichia coli is part of a transmembrane intraprotein pathway conducting protons from the cytoplasm to the heme b595/heme d active site.

Cytochrome bd is a terminal quinol:O 2 oxidoreductase of the respiratory chain of Escherichia coli. The enzyme generates protonmotive force without proton pumping and contains three hemes, b 558, b 595, and d. A highly conserved glutamic acid residue of transmembrane helix III in subunit I, E107, was suggested to be part of a transmembrane pathway delivering protons from the cytoplasm to the oxygen-reducing site. When E107 is replaced with leucine, the hemes are retained but the ubiquinol-1-oxidase activity is lost. We compared wild-type and E107L mutant enzymes during single turnover using absorption and electrometric techniques with a microsecond time resolution. Both wild-type and E107L mutant cytochromes bd in the fully reduced state bind O 2 rapidly, but the formation of the oxoferryl species in the mutant is dramatically retarded as compared to the wild type. Intraprotein electron redistribution induced by the photolysis of CO bound to ferrous heme d in the one-electron-reduced wild-type enzyme is coupled to the membrane potential generation, whereas the mutant cytochrome bd shows no such potential generation. The E107L mutation also causes decrease of midpoint redox potentials of hemes b 595 and d by 25-30 mV and heme b 558 by approximately 70 mV. There are two protonatable groups redox-linked to hemes b 595 and d in the active site, one of which has been recently identified as E445, whereas the second group remains unknown. Here we propose that E107 is either the second group or a key residue of a proposed proton delivery pathway leading from the cytoplasm toward this second group.

Gramicidin S identified as a potent inhibitor for cytochrome bd-type quinol oxidase.

Gramicidin S, a cationic cyclic decapeptide, exhibits the potent antibiotic activity through perturbation of lipid bilayers of the bacterial membrane. From the screening of natural antibiotics, we identified gramicidin S as a potent inhibitor for cytochrome bd-type quinol oxidase from Escherichia coli. We found that gramicidin S inhibited the oxidase with IC(50) of 3.5 microM by decreasing V(max) and the affinity for substrates but showed the stimulatory effect at low concentrations. Our findings would provide a new insight into the development of gramicidin S analogs, which do not share the target and mechanism with conventional antibiotics.

Role of a putative third subunit YhcB on the assembly and function of cytochrome bd-type ubiquinol oxidase from Escherichia coli.

Recent proteome studies on the Escherichia coli membrane proteins suggested that YhcB is a putative third subunit of cytochrome bd-type ubiquinol oxidase (CydAB) (F. Stenberg, P. Chovanec, S.L. Maslen, C.V. Robinson, L.L. Ilag, G. von Heijne, D.O. Daley, Protein complexes of the Escherichia coli cell envelope. J. Biol. Chem. 280 (2005) 34409-34419). We isolated and characterized cytochrome bd from the DeltayhcB strain, and found that the formation of the CydAB heterodimer, the spectroscopic properties of bound hemes, and kinetic parameters for the ubiquinol-1 oxidation were identical to those of cytochrome bd from the wild-type strain. Anion-exchange chromatography and SDS-polyacrylamide gel electrophoresis showed that YhcB was not associated with the cytochrome bd complex. We concluded that YhcB is dispensable for the assembly and function of cytochrome bd. YhcB, which is distributed only in gamma-proteobacteria, may be a part of another membrane protein complex or may form a homo multimeric complex.

Glutamates 99 and 107 in transmembrane helix III of subunit I of cytochrome bd are critical for binding of the heme b595-d binuclear center and enzyme activity.

Cytochrome bd is a quinol oxidase of Escherichia coli under microaerophilic growth conditions. Coupling of the release of protons to the periplasm by quinol oxidation to the uptake of protons from the cytoplasm for dioxygen reduction generates a proton motive force. On the basis of sequence analysis, glutamates 99 and 107 conserved in transmembrane helix III of subunit I have been proposed to convey protons from the cytoplasm to heme d at the periplasmic side. To probe a putative proton channel present in subunit I of E. coli cytochrome bd, we substituted a total of 10 hydrophilic residues and two glycines conserved in helices I and III-V and examined effects of amino acid substitutions on the oxidase activity and bound hemes. We found that Ala or Leu mutants of Arg9 and Thr15 in helix I, Gly93 and Gly100 in helix III, and Ser190 and Thr194 in helix V exhibited the wild-type phenotypes, while Ala and Gln mutants of His126 in helix IV retained all hemes but partially lost the activity. In contrast, substitutions of Thr26 in helix I, Glu99 and Glu107 in helix III, Ser140 in helix IV, and Thr187 in helix V resulted in the concomitant loss of bound heme b558 (T187L) or b595-d (T26L, E99L/A/D, E107L/A/D, and S140A) and the activity. Glu99 and Glu107 mutants except E107L completely lost the heme b595-d center, as reported for heme b595 ligand (His19) mutants. On the basis of this study and previous studies, we propose arrangement of transmembrane helices in subunit I, which may explain possible roles of conserved hydrophilic residues within the membrane.

Kinetic mechanism of quinol oxidation by cytochrome bd studied with ubiquinone-2 analogs.

Cytochrome bd is a heterodimeric terminal ubiquinol oxidase of Escherichia coli under microaerophilic growth conditions. The oxidase activity shows sigmoidal concentration-dependence with low concentrations of ubiquinols, and a marked substrate inhibition with high concentrations of ubiquinol-2 analogs [Sakamoto, K., Miyoshi, H., Takegami, K., Mogi, T., Anraku, Y., and Iwamura H. (1996) J. Biol. Chem. 271, 29897-29902]. Kinetic analysis of the oxidation of the ubiquinol-2 analogs, where the 2- or 3-methoxy group has been substituted with an azido or ethoxy group, suggested that its peculiar enzyme kinetics can be explained by a modified ping-pong bi-bi mechanism with the formation of inactive binary complex FS in the one-electron reduced oxygenated state and inactive ternary complex (E2S)S(n) on the oxidation of the second quinol molecule. Structure-function studies on the ubiquinol-2 analogs suggested that the 6-diprenyl group and the 3-methoxy group on the quinone ring are involved in the substrate inhibition. We also found that oxidized forms of ubiquinone-2 analogs served as weak noncompetitive inhibitors. These results indicate that the mechanism for the substrate oxidation by cytochrome bd is different from that of the heme-copper terminal quinol oxidase and is tightly coupled to dioxygen reduction chemistry.

Cyanide-insensitive quinol oxidase (CIO) from Gluconobacter oxydans is a unique terminal oxidase subfamily of cytochrome bd.

Cyanide-insensitive terminal quinol oxidase (CIO) is a subfamily of cytochrome bd present in bacterial respiratory chain. We purified CIO from the Gluconobacter oxydans membranes and characterized its properties. The air-oxidized CIO showed some or weak peaks of reduced haemes b and of oxygenated and ferric haeme d, differing from cytochrome bd. CO- and NO-binding difference spectra suggested that haeme d serves as the ligand-binding site of CIO. Notably, the purified CIO showed an extraordinary high ubiquinol-1 oxidase activity with the pH optimum of pH 5-6. The apparent Vmax value of CIO was 17-fold higher than that of G. oxydans cytochrome bo3. In addition, compared with Escherichia coli cytochrome bd, the quinol oxidase activity of CIO was much more resistant to cyanide, but sensitive to azide. The Km value for O2 of CIO was 7- to 10-fold larger than that of G. oxydans cytochrome bo3 or E. coli cytochrome bd. Our results suggest that CIO has unique features attributable to the structure and properties of the O2-binding site, and thus forms a new sub-group distinct from cytochrome bd. Furthermore, CIO of acetic acid bacteria may play some specific role for rapid oxidation of substrates under acidic growth conditions.

Diversity in mitochondrial metabolic pathways in parasitic protists Plasmodium and Cryptosporidium.

Apicomplexans are obligate intracellular parasites and occupy diverse niches. They have remodeled mitochondrial carbon and energy metabolism through reductive evolution. Plasmodium lacks mitochondrial pyruvate dehydrogenase and H(+)-translocating NADH dehydrogenase (Complex I, NDH1). The mitochondorion contains a minimal mtDNA ( approximately 6kb) and carries out oxidative phosphorylation in the insect vector stages, by using 2-oxoglutarate as an alternative means of entry into the TCA cycle and a single-subunit flavoprotein as an alternative NADH dehydrogenase (NDH2). In the blood stages of mammalian hosts, mitochondrial enzymes are down-regulated and parasite energy metabolism relies mainly on glycolysis. Mitosomes of Cryptosporidium parvum and Cryptosporidium hominis (human intestine parasites) lack mtDNA, pyruvate dehydrogenase, TCA cycle enzymes except malate-quinone oxidoreductase (MQO), and ATP synthase subunits except alpha and beta. In contrast, mitosomes of Cryptosporidium muris (a rodent gastric parasite) retain all TCA cycle enzymes and functional ATP synthase and carry out oxidative phosphorylation with pyruvate-NADP(+) oxidoreductase (PNO) and a simple and unique respiratory chain consisting of NDH2 and alternative oxidase (AOX). Cryptosporidium and Perkinsus are early branching groups of chromoalveolates (apicomplexa and dinoflagellates, respectively), and both Cryptosporidium mitosome and Perkinsus mitochondrion use PNO, MQO, and AOX. All apicomplexan parasites and dinoflagellates share MQO, which has been acquired from epsilon-proteobacteria via lateral gene transfer. By genome data mining on Plasmodium, Cryptosporidium and Perkinsus, here we summarized their mitochondrial metabolic pathways, which are varied largely from those of mammalian hosts. We hope that our findings will help in understanding the apicomplexan metabolism and development of new chemotherapeutics with novel targets.

Over-expression and characterization of Bacillus subtilis heme O synthase.

Biosynthesis of heme A from heme B is catalysed by two enzymes, heme O and heme A synthases, in the membrane. Heme O synthase in Bacillus subtilis (CtaB) has eight transmembrane helices and catalyses the transfer of a farnesyl group from farnesyl diphosphate to the 2-vinyl group on pyrrole ring A of ferrous heme B. In this study, we constructed the overproduction system for the B. subtilis CtaB in Escherichia coli. We isolated His(7)-CtaB by affinity chromatography and demonstrated the presence of the heme-binding site in heme O synthase. His(7)-CtaB binds substoichiometric amounts of heme B and O, substrate and unreleased product, respectively. Mutagenesis studies suggest that strictly conserved His199 present at the extra-cellular side of helix 5 would serve as the heme-binding site. We are hoping that the overproducing system for heme O synthase would help understanding of detailed mechanism on heme O biosynthesis and X-ray crystallographic studies.

Probing the haem d-binding site in cytochrome bd quinol oxidase by site-directed mutagenesis.

Cytochrome bd is a cyanide-resistant terminal quinol oxidase under micro-aerophilic growth conditions and generates a proton motive force via scalar protolytic reactions. Protons used for dioxygen reduction are taken up from the cytoplasm and delivered to haem d through a proton channel. Electrons are transferred from quinols to haem d through haem b558 and haem b595. All three haems are bound to subunit I but only the axial ligand of haem d remains to be determined. Haems b595 and d form a haem-haem binuclear centre and substitutions of either His19 in helix I (haem b595 ligand) and Glu99 in helix III eliminated or severely reduced both haems. To probe the location of the haem d ligand, we introduced mutations around His19 and Glu99 and examined the cyanide-resistance of the oxidase activity and spectroscopic properties. In contrast to mutations around His19, I98F and L101T reduced the IC50 for cyanide to 0.18 and 0.41 mM, respectively, from 1.4 mM of the wild-type. Blue shifts in the alpha peak of I98F suggest that Ile98 is in the vicinity of the haem d-binding site. Our data are consistent with the proposal that Glu99 serves as a haem d ligand of cytochrome bd.

Probing structure of heme A synthase from Bacillus subtilis by site-directed mutagenesis.

Biosynthesis of heme A from heme B is catalysed by two enzymes, heme O and heme A synthases, in the membrane. Heme A synthase in Bacillus subtilis (CtaA) has eight transmembrane helices and oxidizes a methyl group on pyrrole ring D of heme O to an aldehyde. In this study, to explore structure of heme binding site(s) in heme A synthase, we overproduced the B. subtilis His(6)-CtaA in Escherichia coli and characterized spectroscopic properties of the purified CtaA. On the contrary to a previous report (Svensson, B., Andersson, K.K., and Hederstedt, L. (1996) Low-spin heme A in the heme A biosynthetic protein CtaA from Bacillus subtilis. Eur. J. Biochem. 238, 287-295), we found that two molecules of heme B were bound to CtaA. Further, we demonstrated that substitutions of His60 and His126 did not affect heme binding while His216 and His278 in the carboxy-halves are essential in heme binding. And we found that Ala substitutions of Cys191 and Cys197 in loop 5/6 reduced heme content to a half of the wild-type level. On the basis of our findings, we proposed a helical-wheel-projection model of CtaA.

Effects of replacement of low-spin haem b by haem O on Escherichia coli cytochromes bo and bd quinol oxidases.

Cytochromes bo and bd are terminal ubiquinol oxidases in the aerobic respiratory chain of Escherichia coli and generate proton motive force across the membrane. To probe roles of haem species in the oxidation of quinols, intramolecular electron transfer and the dioxygen reduction, we replaced b-haems with haem O by using the haem O synthase-overproducing system, which can accumulate haem O in cytoplasmic membranes. Characterizations of spectroscopic properties of cytochromes bo and bd isolated from BL21 (DE3)/pLysS and BL21 (DE3)/pLysS/pTTQ18-cyoE after 4 h of the aerobic induction of haem O synthase (CyoE) showed the specific incorporation of haem O into the low-spin haem-binding site in both oxidases. We found that the resultant haem oo- and obd-type oxidase severely reduced the ubiquinol-1 oxidase activity due to the perturbations of the quinol oxidation site. Our observations suggest that haem B is required at the low-spin haem site for the oxidation of quinols by cytochromes bo and bd.