Heme A synthase does not incorporate molecular oxygen into the formyl group of heme A

Heme A is an obligatory cofactor in all eukaryotic and many prokaryotic cytochrome c oxidases. The final step in heme A biosynthesis requires the oxidation of the C8 methyl substituent on pyrrole ring D to an aldehyde, a reaction catalyzed by heme A synthase. To effect this transformation, heme A synthase is proposed to utilize a heme B cofactor, oxidizing the substrate via successive monooxygenase reactions. Consistent with this hypothesis, the activity of heme A synthase is found to be strictly dependent on molecular oxygen. Surprisingly, when cells expressing heme A synthase were incubated with (18)O(2), no significant incorporation of label was observed in heme A, the C8 alcohol intermediate, or the C8 overoxidized byproduct. Conversely, when the cells were grown in H(2)(18)O, partial labeling was observed at every heme oxygen position. These results suggest that the oxygen on the heme A aldehyde is derived from water. Although our data do not allow us to exclude the possibility of exchange with water inside of the cell, the results seem to question a mechanism utilizing successive monooxygenase reactions and support instead a mechanism of heme O oxidation via electron transfer.     

Preimplantation-embryo-specific cell cycle regulation is attributed to the low expression level of retinoblastoma protein

Biosynthesis of heme A, a prosthetic group of cytochrome oxidase (COX), involves an initial farnesylation of heme B. The heme O product formed in this reaction is modified by hydroxylation of the methyl group at carbon C-8 of the porphyrin ring. This reaction was proposed to be catalyzed by Cox15p, ferredoxin, and ferredoxin reductase. Oxidation of the alcohol to the corresponding aldehyde yields heme A. In the present study we have assayed heme A and heme O in yeast COX mutants. The steady state concentrations of the two hemes in the different strains studied indicate that hydroxylation of heme O, catalyzed by Cox15p, is regulated either by a subunit or assembly intermediate of COX. The heme profiles of the mutants also suggest positive regulation of heme B farnesylation by the hydroxylated intermediate formed at the subsequent step or by Cox15p itself.     

Heme O synthase and heme A synthase from Bacillus subtilis and Rhodobacter sphaeroides interact in Escherichia coli

Cytochrome c oxidase requires multiple heme and copper cofactors to catalyze the reduction of molecular oxygen to water. Although significant progress has been made in understanding the transport and incorporation of the copper ions, considerably less is known about the trafficking and insertion of the heme cofactors. Heme O synthase (HOS) and heme A synthase (HAS) from Rhodobacter sphaeroides (Cox10 and Cox15, respectively) and Bacillus subtilis (CtaB and CtaA, respectively) have been cloned and expressed in Escherichia coli. Our results demonstrate that HOS copurifies with HAS and that HAS copurifies with HOS, indicating that HOS and HAS interact and may form a physiologically relevant complex in vivo. Consistent with this hypothesis, the presence of HAS alters the total level of farnesylated hemes, providing further evidence that HOS and HAS interact. Our current working model is that HOS and HAS form a complex and that heme O is transferred directly from HOS to HAS. Because of the strong sequence similarity and evolutionary relationship between R. sphaeroides and mitochondria, our data suggest that this complex may form in eukaryotes as well.     

The low-spin heme site of cytochrome o from Escherichia coli is promiscuous with respect to heme type.

Cytochrome o of Escherichia coli is able to incorporate two different structures of heme, either heme B (protoheme) or heme O, in its low-spin heme site. In contrast, the heme of the binuclear O2 reduction site is invariably heme O. Heme O is a newly discovered heme that is related to heme A, but with the formyl group of the latter replaced by methyl. Enzyme isolated from wild type E. coli has predominantly heme B in the low-spin site, whereas enzyme isolated from various overexpressing strains contains both types of enzyme in different proportions. In some strains, 70% of the enzyme has heme O in the low-spin site. Despite this variation in the structure of one of the prosthetic groups, the enzymatic activity and polypeptide composition of the enzyme remain virtually constant. EPR and activity data both indicate that heme B and heme O occupy the same low-spin heme site in the enzyme. With heme O in this site, the alpha-absorption band is narrower and further to the blue, and the Em,7 is lower, than when there is heme B in the site. In contrast to previous proposals, we show here that the enzyme does not exhibit significant spectral interactions between the hemes. The structural heterogeneity of the low-spin heme accounts for the variation in the optical spectra and redox properties of the enzyme as isolated from different strains of E. coli.     

Heme A biosynthesis

Respiration in plants, most animals and many aerobic microbes is dependent on heme A. This is a highly specialized type of heme found as prosthetic group in cytochrome a-containing respiratory oxidases. Heme A differs structurally from heme B (protoheme IX) by the presence of a hydroxyethylfarnesyl group instead of a vinyl side group at the C2 position and a formyl group instead of a methyl side group at position C8 of the porphyrin macrocycle. Heme A synthase catalyzes the formation of the formyl side group and is a poorly understood heme-containing membrane bound atypical monooxygenase. This review presents our current understanding of heme A synthesis at the molecular level in mitochondria and aerobic bacteria. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.     

The Bacillus subtilis ctaB paralogue, yjdK, can complement the heme A synthesis deficiency of a CtaB-deficient mutant

Heme A is a prosthetic group in many respiratory oxidases. It is synthesised from heme B (protoheme IX) with heme O as an intermediate. In Bacillus subtilis two genes required for heme A synthesis, ctaA and ctaB, have been identified. CtaB is the heme O synthase and CtaA is involved in the conversion of heme O to heme A. A ctaB paralogue, yjdK, has been identified through the B. subtilis genome sequencing project. In this study we show that when carried on a low copy number plasmid, the yjdK gene can complement a ctaB deletion mutant with respect to heme A synthesis. Our results indicate that YjdK has heme O synthase activity. We therefore suggest that yjdK be renamed as ctaO.     

Heme A biosynthesis

Respiration in plants, most animals and many aerobic microbes is dependent on heme A. This is a highly specialized type of heme found as prosthetic group in cytochrome a-containing respiratory oxidases. Heme A differs structurally from heme B (protoheme IX) by the presence of a hydroxyethylfarnesyl group instead of a vinyl side group at the C2 position and a formyl group instead of a methyl side group at position C8 of the porphyrin macrocycle. Heme A synthase catalyzes the formation of the formyl side group and is a poorly understood heme-containing membrane bound atypical monooxygenase. This review presents our current understanding of heme A synthesis at the molecular level in mitochondria and aerobic bacteria. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.     

Glutamate 107 in subunit I of the cytochrome bd quinol oxidase from Escherichia coli is protonated and near the heme d/heme b595 binuclear center

Cytochrome bd is a quinol oxidase from Escherichia coli, which is optimally expressed under microaerophilic growth conditions. The enzyme catalyzes the two-electron oxidation of either ubiquinol or menaquinol in the membrane and scavenges O2 at low concentrations, reducing it to water. Previous work has shown that, although cytochrome bd does not pump protons, turnover is coupled to the generation of a proton motive force. The generation of a proton electrochemical gradient results from the release of protons from the oxidation of quinol to the periplasm and the uptake of protons used to form H2O from the cytoplasm. Because the active site has been shown to be located near the periplasmic side of the membrane, a proton channel must facilitate the delivery of protons from the cytoplasm to the site of water formation. Two conserved glutamic acid residues, E107 and E99, are located in transmembrane helix III in subunit I and have been proposed to form part of this putative proton channel. In the current work, it is shown that mutations in either of these residues results in the loss of quinol oxidase activity and can result in the loss of the two hemes at the active site, hemes d and b595. One mutant, E107Q, while being totally inactive, retains the hemes. Fourier transform infrared (FTIR) redox difference spectroscopy has identified absorption bands from the COOH group of E107. The data show that E107 is protonated at pH 7.6 and that it is perturbed by the reduction of the heme d/heme b595 binuclear center at the active site. In contrast, mutation of an acidic residue known to be at or near the quinol-binding site (E257A) also inactivates the enzyme but has no substantial influence on the FTIR redox difference spectrum. Mutagenesis shows that there are several acidic residues, including E99 and E107 as well as D29 (in CydB), which are important for the assembly or stability of the heme d/heme b595 active site.     

Properties of the two terminal oxidases of Escherichia coli.

Proton translocation coupled to oxidation of ubiquinol by O2 was studied in spheroplasts of two mutant strains of Escherichia coli, one of which expresses cytochrome d, but not cytochrome bo, and the other expressing only the latter. O2 pulse experiments revealed that cytochrome d catalyzes separation of the protons and electrons of ubiquinol oxidation but is not a proton pump. In contrast, cytochrome bo functions as a proton pump in addition to separating the charges of quinol oxidation. E. coli membranes and isolated cytochrome bo lack the CuA center typical of cytochrome c oxidase, and the isolated enzyme contains only 1Cu/2Fe. Optical spectra indicate that high-spin heme o contributes less than 10% to the reduced minus oxidized 560-nm band of the enzyme. Pyridine hemochrome spectra suggest that the hemes of cytochrome bo are not protohemes. Proteoliposomes with cytochrome bo exhibited good respiratory control, but H+/e- during quinol oxidation was only 0.3-0.7. This was attributed to an "inside out" orientation of a significant fraction of the enzyme. Possible metabolic benefits of expressing both cytochromes bo and d in E. coli are discussed.     

Heme P460 of hydroxylamine oxidoreductase of Nitrosomonas. Reaction with CO and H2O2

Hydroxylamine oxidoreductase (HAO) of Nitrosomonas catalyzes the dehydrogenation of NH2OH and subsequent addition of oxygen to form nitrite. HAO contains c hemes and the CO-binding heme P460 in a 7:1 ratio; dehydrogenation of NH2OH involves passage of electrons to P460 and then c hemes. We now report that electrons rapidly pass from c hemes of HAO to the P460 center and then to H2O2. This conclusion is supported by (a) inhibition of c heme oxidation with CO and (b) loss of H2O2-oxidizability of ferrous c hemes following specific destruction of heme P460. Reaction of ferrous P460 with H2O2 is rate-limiting. Activation of dioxygen for N-oxidation by ferrous HAO may involve the two-electron reduction of O2 by P460. The reaction of ferrous HAO with H2O2 was studied as it may reveal aspects of the mechanism of activation of dioxygen. Reaction of ferrous heme P460 with CO is slow and with low affinity as compared with other hemoproteins. Values for reaction of CO with enzyme were: k1, 1.1 X 10(-3) M-1 s-1 and Kd, 12 microM.     

Heme A synthase enzyme functions dissected by mutagenesis of Bacillus subtilis CtaA

Heme A, as a prosthetic group, is found exclusively in respiratory oxidases of mitochondria and aerobic bacteria. Bacillus subtilis CtaA and other heme A synthases catalyze the conversion of a methyl side group on heme O into a formyl group. The catalytic mechanism of heme A synthase is not understood, and little is known about the composition and structure of the enzyme. In this work, we have: (i) constructed a ctaA deletion mutant and a system for overproduction of mutant variants of the CtaA protein in B. subtilis, (ii) developed anaffinity purification procedure for isolation of preparative amounts of CtaA, and (iii) investigated the functional roles of four invariant histidine residues in heme A synthase by in vivo and in vitro analyses of the properties of mutant variants of CtaA. Our results show an important function of three histidine residues for heme A synthase activity. Several of the purified mutant enzyme proteins contained tightly bound heme O. One variant also contained trapped hydroxylated heme O, which is a postulated enzyme reaction intermediate. The findings indicate functional roles for the invariant histidine residues and provide strong evidence that the heme A synthase enzyme reaction includes two consecutive monooxygenations.     

Mechanism of horseradish peroxidase-catalyzed heme oxidation and polymerization (beta-hematin formation)

Horseradish peroxidase (HRP) catalyzes the polymerization of free heme (beta-hematin formation) through its oxidation. Heme when added to HRP compound II (FeIV=O) causes spectral shift from 417 nm (Compound II) to 402 nm (native, FeIII) indicating that heme may be oxidized via one-electron transfer. Direct evidence for one-electron oxidation of heme by HRP intermediates is provided by the appearance of an E.s.r signal of a 5,5-dimethyl-1-pyrroline N-oxide (spin trap)-heme radical adduct (a1H=14.75 G, a2H=4.0 G) in E.s.r studies. Heme-polymerization by HRP is inhibited by spin trap indicating that one-electron oxidation product of heme ultimately leads to the formation of heme-polymer. HRP, when incubated with diethyl pyrocarbonate (DEPC), a histidine specific reagent, shows concentration dependent loss of heme-polymerization indicating the role of histidine residues in the process. We suggest that HRP catalyzes the formation of heme-polymer through one-electron oxidation of free heme.     

Evidence for two different electron transfer pathways in the same enzyme, nitrate reductase A from Escherichia coli.

In order to clarify the role of cytochrome in nitrate reductase we have performed spectrophotometric and stopped-flow kinetic studies of reduction and oxidation of the cytochrome hemes with analogues of physiological quinones, using menadione as an analogue of menaquinone and duroquinone as an analogue of ubiquinone, and comparing the results with those obtained with dithionite. The spectrophotometric studies indicate that reduction of the cytochrome hemes varies according to the analogue of quinone used, and in no cases is it complete. Stopped-flow kinetics of heme oxidation by potassium nitrate indicates that there are two distinct reactions, depending on whether the hemes were previously reduced by menadiol or by duroquinol. These results, and those of spectrophotometric studies of a mutant lacking the highest-potential [Fe-S] cluster, allow us to propose a two-pathway electron transfer model for nitrate reductase A from Escherichia coli.     

Bacillus subtilis CtaA is a heme-containing membrane protein involved in heme A biosynthesis.

Heme A is a prosthetic group of many respiratory oxidases. It is synthesized from protoheme IX (heme B) seemingly with heme O as a stable intermediate. The Bacillus subtilis ctaA and ctaB genes are required for heme A and heme O synthesis, respectively (B. Svensson, M. Lübben, and L. Hederstedt, Mol. Microbiol. 10:193-201, 1993). Tentatively, CtaA is involved in the monooxygenation and oxidation of the methyl side group on porphyrin ring D in heme A synthesis from heme B. B. subtilis ctaA and ctaB on plasmids in both B. subtilis and Escherichia coli were found to result in a novel membrane-bound heme-containing protein with the characteristics of a low-spin b-type cytochrome. It can be reduced via the respiratory chain, and in the reduced state it shows light absorption maxima at 428, 528, and 558 nm and the alpha-band is split. Purified cytochrome isolated from both B. subtilis and E. coli membranes contained one polypeptide identified as CtaA by amino acid sequence analysis, about 0.2 mol of heme B per mol of polypeptide, and small amounts of heme A.     

Tryptophan tryptophylquinone biosynthesis: a radical approach to posttranslational modification

Protein-derived cofactors are formed by irreversible covalent posttranslational modification of amino acid residues. An example is tryptophan tryptophylquinone (TTQ) found in the enzyme methylamine dehydrogenase (MADH). TTQ biosynthesis requires the cross-linking of the indole rings of two Trp residues and the insertion of two oxygen atoms onto adjacent carbons of one of the indole rings. The diheme enzyme MauG catalyzes the completion of TTQ within a precursor protein of MADH. The preMADH substrate contains a single hydroxyl group on one of the tryptophans and no crosslink. MauG catalyzes a six-electron oxidation that completes TTQ assembly and generates fully active MADH. These oxidation reactions proceed via a high valent bis-Fe(IV) state in which one heme is present as Fe(IV)=O and the other is Fe(IV) with both axial heme ligands provided by amino acid side chains. The crystal structure of MauG in complex with preMADH revealed that catalysis does not involve direct contact between the hemes of MauG and the protein substrate. Rather it is accomplished through long-range electron transfer, which presumably generates radical intermediates. Kinetic, spectrophotometric, and site-directed mutagenesis studies are beginning to elucidate how the MauG protein controls the reactivity of the hemes and mediates the long range electron/radical transfer required for catalysis. This article is part of a Special Issue entitled: Radical SAM enzymes and Radical Enzymology.     

Stereoselectivity of each of the three steps of the heme oxygenase reaction: hemin to meso-hydroxyhemin,meso-hydroxyhemin to verdoheme,and verdoheme to biliverdin

Heme oxygenase catalyzes the regiospecific oxidation of hemin to biliverdin IXalpha with concomitant liberation of CO and iron by three sequential monooxygenase reactions. The alpha-regioselectivity of heme oxygenase has been thought to result from the regioselective oxygenation of the heme alpha-meso position at the first step, which leads to the reaction pathway via meso-hydroxyheme IXalpha and verdoheme IXalpha intermediates. However, recent reports concerning heme oxygenase forming biliverdin isomers other than biliverdin IXalpha raise a question whether heme oxygenase can degrade meso-hydroxyhemin and isomers other than the alpha-isomers. In this paper, we investigated the stereoselectivity of each of the two reaction steps from meso-hydroxyhemin to verdoheme and verdoheme to biliverdin by using a truncated form of rat heme oxygenase-1 and the chemically synthesized four isomers of meso-hydroxyhemin and verdoheme. Heme oxygenase-1 converted all four isomers of meso-hydroxyhemin to the corresponding isomers of verdoheme. In contrast, only verdoheme IXalpha was converted to the corresponding biliverdin IXalpha. We conclude that the third step, but not the second, is stereoselective for the alpha-isomer substrate. The present findings on regioselectivities of the second and the third steps have been discussed on the basis of the oxygen activation mechanisms of these steps.     

X-ray crystallographic and biochemical characterization of the inhibitory action of an imidazole-dioxolane compound on heme oxygenase

Heme oxygenase (HO) catalyzes the regiospecific cleavage of the porphyrin ring of heme using reducing equivalents and O2 to produce biliverdin, iron, and CO. Because CO has a cytoprotective effect through the p38-MAPK pathway, HO is a potential therapeutic target in cancer. In fact, inhibition of the HO isoform HO-1 reduces Kaposi sarcoma tumor growth. Imidazole-dioxolane compounds have recently attracted attention because they have been reported to specifically inhibit HO-1, but not HO-2, unlike Cr-containing protoporphyrin IX, a classical inhibitor of HO, that inhibits not only both HO isoforms but also other hemoproteins. The inhibitory mechanism of imidazole-dioxolane compounds, however, has not yet been characterized. Here, we determine the crystal structure of the ternary complex of rat HO-1, heme, and an imidazole-dioxolane compound, 2-[2-(4-chlorophenyl)ethyl]-2-[(1H-imidazol-1-yl)methyl]-1,3-dioxolane. This compound bound on the distal side of the heme iron, where the imidazole and 4-chlorophenyl groups were bound to the heme iron and the hydrophobic cavity in HO, respectively. Binding of the bulky inhibitor in the narrow distal pocket shifted the distal helix to open the distal site and moved both the heme and the proximal helix. Furthermore, the biochemical characterization revealed that the catalytic reactions of both HO-1 and HO-2 were completely stopped after the formation of verdoheme in the presence of the imidazole-dioxolane compound. This result should be mainly due to the lower reactivity of the inhibitor-bound verdoheme with O2 compared to the reactivity of the inhibitor-bound heme with O2.     

Functional importance of tyrosine 294 and the catalytic selectivity for the bis-Fe(IV) state of MauG revealed by replacement of this axial heme ligand with histidine

The diheme enzyme MauG catalyzes the posttranslational modification of a precursor protein of methylamine dehydrogenase (preMADH) to complete the biosynthesis of its protein-derived tryptophan tryptophylquinone (TTQ) cofactor. It catalyzes three sequential two-electron oxidation reactions which proceed through a high-valent bis-Fe(IV) redox state. Tyr294, the unusual distal axial ligand of one c-type heme, was mutated to His, and the crystal structure of Y294H MauG in complex with preMADH reveals that this heme now has His-His axial ligation. Y294H MauG is able to interact with preMADH and participate in interprotein electron transfer, but it is unable to catalyze the TTQ biosynthesis reactions that require the bis-Fe(IV) state. This mutation affects not only the redox properties of the six-coordinate heme but also the redox and CO-binding properties of the five-coordinate heme, despite the 21 ? separation of the heme iron centers. This highlights the communication between the hemes which in wild-type MauG behave as a single diheme unit. Spectroscopic data suggest that Y294H MauG can stabilize a high-valent redox state equivalent to Fe(V), but it appears to be an Fe(IV)═O/π radical at the five-coordinate heme rather than the bis-Fe(IV) state. This compound I-like intermediate does not catalyze TTQ biosynthesis, demonstrating that the bis-Fe(IV) state, which is stabilized by Tyr294, is specifically required for this reaction. The TTQ biosynthetic reactions catalyzed by wild-type MauG do not occur via direct contact with the Fe(IV)═O heme but via long-range electron transfer through the six-coordinate heme. Thus, a critical feature of the bis-Fe(IV) species may be that it shortens the electron transfer distance from preMADH to a high-valent heme iron.     

Mitochondrial ferredoxin is required for heme A synthesis in Saccharomyces cerevisiae.

Heme A is a prosthetic group of all eukaryotic and some prokaryotic cytochrome oxidases. This heme differs from heme B (protoheme) at two carbon positions of the porphyrin ring. The synthesis of heme A begins with farnesylation of the vinyl group at carbon C-2 of heme B. The heme O product of this reaction is then converted to heme A by a further oxidation of a methyl to a formyl group on C-8. In a previous study (Barros, M. H., Carlson, C. G., Glerum, D. M., and Tzagoloff, A. (2001) FEBS Lett. 492, 133-138) we proposed that the formyl group is formed by an initial hydroxylation of the C-8 methyl by a three-component monooxygenase consisting of Cox15p, ferredoxin, and ferredoxin reductase. In the present study three lines of evidence confirm a requirement of ferredoxin in heme A synthesis. 1) Temperature-conditional yah1 mutants grown under restrictive conditions display a decrease in heme A relative to heme B. 2) The incorporation of radioactive delta-aminolevulinic acid into heme A is reduced in yah1 ts but not in the wild type after the shift to the restrictive temperature; and 3) the overexpression of Cox15p in cytochrome oxidase mutants that accumulate heme O leads to an increased mitochondrial concentration of heme A. The increase in heme A is greater in mutants that overexpress Cox15p and ferredoxin. These results are consistent with a requirement of ferredoxin and indirectly of ferredoxin reductase in hydroxylation of heme O.