Catharina T. Migita

Anammox organism KSU-1 expresses a NirK-type copper-containing nitrite reductase instead of a NirS-type with cytochrome cd1.

Anaerobic ammonium oxidation (anammox) and denitrification are two distinct microbial reactions relevant to the global nitrogen cycle. The proposed initial step of the anammox reactions, reduction of nitrite to nitric oxide, has been postulated to be identical to that in denitrification catalyzed by the dissimilatory nitrite reductase of the cytochrome cd 1‐type. Here, we characterized the copper‐containing nitrite reductase homolog encoded by nirK detected in the genome of an anammox bacterium strain KSU‐1. We hypothesize that this NirK‐type nitrite reductase, rather than a nitrite reductase of the cytochrome cd 1‐type (NirS), is likely to catalyze nitrite reduction in anammox organism KSU‐1.

The Oxygen and Carbon Monoxide Reactions of Heme Oxygenase

The O2 and CO reactions with the heme, α-hydroxyheme, and verdoheme complexes of heme oxygenase have been studied. The heme complexes of heme oxygenase isoforms-1 and -2 have similar O2 and CO binding properties. The O2 affinities are very high,K O2 = 30–80 μm −1, which is 30–90-fold greater than those of mammalian myoglobins. The O2 association rate constants are similar to those for myoglobins (k O2 ′ = 7–20 μm −1 s−1), whereas the O2 dissociation rates are remarkably slow (k O2 = 0.25 s−1), implying the presence of very favorable interactions between bound O2 and protein residues in the heme pocket. The CO affinities estimated for both isoforms are only 1–6-fold higher than the corresponding O2 affinities. Thus, heme oxygenase discriminates much more strongly against CO binding than either myoglobin or hemoglobin. The CO binding reactions with the ferrous α-hydroxyheme complex are similar to those of the protoheme complex, and hydroxylation at the α-meso position does not appear to affect the reactivity of the iron atom. In contrast, the CO affinities of the verdoheme complexes are >10,000 times weaker than those of the heme complexes because of a 100-fold slower association rate constant (k CO′   ≈ 0.004 μm −1 s−1) and a 300-fold greater dissociation rate constant (k CO ≈ 3 s−1) compared with the corresponding rate constants of the protoheme and α-hydroxyheme complexes. The positive charge on the verdoporphyrin ring causes a large decrease in reactivity of the iron.

Escherichia coli PQQ-containing quinoprotein glucose dehydrogenase: its structure comparison with other quinoproteins.

Membrane-bound glucose dehydrogenase (mGDH) in Escherichia coli is one of the pivotal pyrroloquinoline quinone (PQQ)-containing quinoproteins coupled with the respiratory chain in the periplasmic oxidation of alcohols and sugars in Gram-negative bacteria. We compared mGDH with other PQQ-dependent quinoproteins in molecular structure and attempted to trace their evolutionary process. We also review the role of residues crucial for the catalytic reaction or for interacting with PQQ and discuss the functions of two distinct domains, radical formation in PQQ, and the presumed existence of bound quinone in mGDH.

O2- and H2O2-dependent Verdoheme Degradation by Heme Oxygenase REACTION MECHANISMS AND POTENTIAL PHYSIOLOGICAL ROLES OF THE DUAL PATHWAY DEGRADATION

Heme oxygenase (HO) catalyzes the catabolism of heme to biliverdin, CO, and a free iron through three successive oxygenation steps. The third oxygenation, oxidative degradation of verdoheme to biliverdin, has been the least understood step despite its importance in regulating HO activity. We have examined in detail the degradation of a synthetic verdoheme IXα complexed with rat HO-1. Our findings include: 1) HO degrades verdoheme through a dual pathway using either O2 or H2O2; 2) the verdoheme reactivity with O2 is the lowest among the three O2 reactions in the HO catalysis, and the newly found H2O2 pathway is ∼40-fold faster than the O2-dependent verdoheme degradation; 3) both reactions are initiated by the binding of O2 or H2O2 to allow the first direct observation of degradation intermediates of verdoheme; and 4) Asp140 in HO-1 is critical for the verdoheme degradation regardless of the oxygen source. On the basis of these findings, we propose that the HO enzyme activates O2 and H2O2 on the verdoheme iron with the aid of a nearby water molecule linked with Asp140. These mechanisms are similar to the well established mechanism of the first oxygenation, meso-hydroxylation of heme, and thus, HO can utilize a common architecture to promote the first and third oxygenation steps of the heme catabolism. In addition, our results infer the possible involvement of the H2O2-dependent verdoheme degradation in vivo, and potential roles of the dual pathway reaction of HO against oxidative stress are proposed.

Occurrence of a bound ubiquinone and its function in Escherichia coli membrane-bound quinoprotein glucose dehydrogenase.

The membrane-bound pyrroloquinoline quinone (PQQ)-containing quinoprotein glucose dehydrogenase (mGDH) in Escherichia coli functions by catalyzing glucose oxidation in the periplasm and by transferring electrons directly to ubiquinone (UQ) in the respiratory chain. To clarify the intramolecular electron transfer of mGDH, quantitation and identification of UQ were performed, indicating that purified mGDH contains a tightly bound UQ8 in its molecule. A significant increase in the EPR signal was observed following glucose addition in mGDH reconstituted with PQQ and Mg2+, suggesting that bound UQ8 accepts a single electron from PQQH2 to generate semiquinone radicals. No such increase in the EPR signal was observed in UQ8-free mGDH under the same conditions. Moreover, a UQ2 reductase assay with a UQ-related inhibitor (C49) revealed different inhibition kinetics between the wild-type mGDH and UQ8-free mGDH. From these findings, we propose that the native mGDH bears two ubiquinone-binding sites, one (QI) for bound UQ8 in its molecule and the other (QII) for UQ8 in the ubiquinone pool, and that the bound UQ8 in the QI site acts as a single electron mediator in the intramolecular electron transfer in mGDH.

Electron Transfer Ability from NADH to Menaquinone and from NADPH to Oxygen of Type II NADH Dehydrogenase of Corynebacterium glutamicum

Type II NADH dehydrogenase of Corynebacterium glutamicum (NDH-2) was purified from an ndh overexpressing strain. Purification conferred 6-fold higher specific activity of NADH:ubiquinone-1 oxidoreductase with a 3.5-fold higher recovery than that previously reported (K. Matsushita et al., 2000). UV–visible and fluorescence analyses of the purified enzyme showed that NDH-2 of C. glutamicum contained non-covalently bound FAD but not covalently bound FMN. This enzyme had an ability to catalyze electron transfer from NADH and NADPH to oxygen as well as various artificial quinone analogs at neutral and acidic pHs respectively. The reduction of native quinone of C. glutamicum, menaquinone-2, with this enzyme was observed only with NADH, whereas electron transfer to oxygen was observed more intensively with NADPH. This study provides evidence that C. glutamicum NDH-2 is a source of the reactive oxygen species, superoxide and hydrogen peroxide, concomitant with NADH and NADPH oxidation, but especially with NADPH oxidation. Together with this unique character of NADPH oxidation, phylogenetic analysis of NDH-2 from various organisms suggests that NDH-2 of C. glutamicum is more closely related to yeast or fungal enzymes than to other prokaryotic enzymes.

Protein expressed by the ho2 gene of the cyanobacterium Synechocystis sp. PCC 6803 is a true heme oxygenase. Properties of the heme and enzyme complex.

Two isoforms of a heme oxygenase gene, ho1 and ho2, with 51% identity in amino acid sequence have been identified in the cyanobacterium Synechocystis sp. PCC 6803. Isoform‐1, Syn HO‐1, has been characterized, while isoform‐2, Syn HO‐2, has not. In this study, a full‐length ho2 gene was cloned using synthetic DNA and Syn HO‐2 was demonstrated to be highly expressed in Escherichia coli as a soluble, catalytically active protein. Like Syn HO‐1, the purified Syn HO‐2 bound hemin stoichiometrically to form a heme–enzyme complex and degraded heme to biliverdin IXα, CO and iron in the presence of reducing systems such as NADPH/ferredoxin reductase/ferredoxin and sodium ascorbate. The activity of Syn HO‐2 was found to be comparable to that of Syn HO‐1 by measuring the amount of bilirubin formed. In the reaction with hydrogen peroxide, Syn HO‐2 converted heme to verdoheme. This shows that during the conversion of hemin to α‐meso‐hydroxyhemin, hydroperoxo species is the activated oxygen species as in other heme oxygenase reactions. The absorption spectrum of the hemin–Syn HO‐2 complex at neutral pH showed a Soret band at 412 nm and two peaks at 540 nm and 575 nm, features observed in the hemin‐Syn HO‐1 complex at alkaline pH, suggesting that the major species of iron(III) heme iron at neutral pH is a hexa‐coordinate low spin species. Electron paramagnetic resonance (EPR) revealed that the iron(III) complex was in dynamic equilibrium between low spin and high spin states, which might be caused by the hydrogen bonding interaction between the distal water ligand and distal helix components. These observations suggest that the structure of the heme pocket of the Syn HO‐2 is different from that of Syn HO‐1.

Substrate binding-induced changes in the EPR spectra of the ferrous nitric oxide complexes of neuronal nitric oxide synthase.

A versatile diatomic physiological messenger, nitric oxide (NO), is biosynthesized by a group of flavo-heme enzymes, the nitric oxide synthases. We have examined the active site of the neuronal isoform by EPR spectroscopy of the ferrous nitric oxide complex. The nitric oxide complex of the substrate-free enzyme exhibits a cytochrome P450-type EPR spectrum typical of a hexacoordinate NO-heme complex with a non-nitrogenous proximal axial heme ligand. The NO complex of the substrate-free enzyme is rather unstable and spontaneously converts to a cytochrome P420 type pentacoordinate denatured form. Binding of L-arginine (l-Arg) enhances the stability of the hexacoordinate NO form. The EPR spectrum of the NO adduct of the enzyme-substrate complex has an increased g-anisotropy and well-resolved hyperfine couplings due to the 14N of nitric oxide. Significant perturbations in the NO EPR spectrum were observed upon Nomega-monomethyl-L-Arg and Nomega-hydroxy-L-Arg binding. The perturbations in the EPR spectrum indicate that L-Arg and its derivatives bind on the distal site of the heme in very close proximity to the bound NO to cause alterations in the heme-NO coordination structure. Interactions between the bound NO and the substrate or its analogues appear to affect the Fe-NO geometry, resulting in the observed spectral changes. We infer that analogous interactions with oxygen might be involved in the hydroxylation events during enzyme catalysis of nitric oxide synthase.

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.

Menaquinone as Well as Ubiquinone as a Bound Quinone Crucial for Catalytic Activity and Intramolecular Electron Transfer in Escherichia coli Membrane-bound Glucose Dehydrogenase

Escherichia coli membrane-bound glucose dehydrogenase (mGDH), which is one of quinoproteins containing pyrroloquinoline quinone (PQQ) as a coenzyme, is a good model for elucidating the function of bound quinone inside primary dehydrogenases in respiratory chains. Enzymatic analysis of purified mGDH from cells defective in synthesis of ubiquinone (UQ) and/or menaquinone (MQ) revealed that Q-free mGDH has very low levels of activity of glucose dehydrogenase and UQ2 reductase compared with those of UQ-bearing mGDH, and both activities were significantly increased by reconstitution with UQ1. On the other hand, MQ-bearing mGDH retains both catalytic abilities at the same levels as those of UQ-bearing mGDH. A radiolytically generated hydrated electron reacted with the bound MQ to form a semiquinone anion radical with an absorption maximum at 400 nm. Subsequently, decay of the absorbance at 400 nm was accompanied by an increase in the absorbance at 380 nm with a first order rate constant of 5.7 × 103 s–1. This indicated that an intramolecular electron transfer from the bound MQ to the PQQ occurred. EPR analysis revealed that characteristics of the semiquinone radical of bound MQ are similar to those of the semiquinone radical of bound UQ and indicated an electron flow from PQQ to MQ as in the case of UQ. Taken together, the results suggest that MQ is incorporated into the same pocket as that for UQ to perform a function almost equivalent to that of UQ and that bound quinone is involved at least partially in the catalytic reaction and primarily in the intramolecular electron transfer of mGDH.