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Characterization of a nitrite-reducing octaheme hydroxylamine oxidoreductase that lacks the tyrosine cross-link

2021; Elsevier BV; Volume: 296; Linguagem: Inglês

10.1016/j.jbc.2021.100476

ISSN

1083-351X

Autores

Christina Ferousi, Rob A. Schmitz, Wouter J. Maalcke, Simon Lindhoud, Wouter Versantvoort, Mike S. M. Jetten, Joachim Reimann, Boran Kartal,

Tópico(s)

Heme Oxygenase-1 and Carbon Monoxide

Resumo

The hydroxylamine oxidoreductase (HAO) family consists of octaheme proteins that harbor seven bis-His ligated electron-transferring hemes and one 5-coordinate catalytic heme with His axial ligation. Oxidative HAOs have a homotrimeric configuration with the monomers covalently attached to each other via a unique double cross-link between a Tyr residue and the catalytic heme moiety of an adjacent subunit. This cross-linked active site heme, termed the P460 cofactor, has been hypothesized to modulate enzyme reactivity toward oxidative catalysis. Conversely, the absence of this cross-link is predicted to favor reductive catalysis. However, this prediction has not been directly tested. In this study, an HAO homolog that lacks the heme-Tyr cross-link (HAOr) was purified to homogeneity from the nitrite-dependent anaerobic ammonium-oxidizing (anammox) bacterium Kuenenia stuttgartiensis, and its catalytic and spectroscopic properties were assessed. We show that HAOr reduced nitrite to nitric oxide and also reduced nitric oxide and hydroxylamine as nonphysiological substrates. In contrast, HAOr was not able to oxidize hydroxylamine or hydrazine supporting the notion that cross-link-deficient HAO enzymes are reductases. Compared with oxidative HAOs, we found that HAOr harbors an active site heme with a higher (at least 80 mV) midpoint potential and a much lower degree of porphyrin ruffling. Based on the physiology of anammox bacteria and our results, we propose that HAOr reduces nitrite to nitric oxide in vivo, providing anammox bacteria with NO, which they use to activate ammonium in the absence of oxygen. The hydroxylamine oxidoreductase (HAO) family consists of octaheme proteins that harbor seven bis-His ligated electron-transferring hemes and one 5-coordinate catalytic heme with His axial ligation. Oxidative HAOs have a homotrimeric configuration with the monomers covalently attached to each other via a unique double cross-link between a Tyr residue and the catalytic heme moiety of an adjacent subunit. This cross-linked active site heme, termed the P460 cofactor, has been hypothesized to modulate enzyme reactivity toward oxidative catalysis. Conversely, the absence of this cross-link is predicted to favor reductive catalysis. However, this prediction has not been directly tested. In this study, an HAO homolog that lacks the heme-Tyr cross-link (HAOr) was purified to homogeneity from the nitrite-dependent anaerobic ammonium-oxidizing (anammox) bacterium Kuenenia stuttgartiensis, and its catalytic and spectroscopic properties were assessed. We show that HAOr reduced nitrite to nitric oxide and also reduced nitric oxide and hydroxylamine as nonphysiological substrates. In contrast, HAOr was not able to oxidize hydroxylamine or hydrazine supporting the notion that cross-link-deficient HAO enzymes are reductases. Compared with oxidative HAOs, we found that HAOr harbors an active site heme with a higher (at least 80 mV) midpoint potential and a much lower degree of porphyrin ruffling. Based on the physiology of anammox bacteria and our results, we propose that HAOr reduces nitrite to nitric oxide in vivo, providing anammox bacteria with NO, which they use to activate ammonium in the absence of oxygen. Cytochrome c proteins are among the most common redox tools in nature and are ubiquitous in all domains of life (1Richardson D.J. Bacterial respiration: A flexible process for a changing environment.Microbiology. 2000; 146: 551-571Crossref PubMed Scopus (289) Google Scholar). 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Structure and sequence conservation of hao cluster genes of autotrophic ammonia-oxidizing bacteria: Evidence for their evolutionary history.Appl. Environ. Microbiol. 2005; 71: 5371-5382Crossref PubMed Scopus (58) Google Scholar). The HAO family comprises enzymes that harbor eight CX2–4CH heme-binding motifs, but otherwise exhibits low primary structure similarity (10Bergmann D.J. Hooper A.B. Klotz M.G. Structure and sequence conservation of hao cluster genes of autotrophic ammonia-oxidizing bacteria: Evidence for their evolutionary history.Appl. Environ. Microbiol. 2005; 71: 5371-5382Crossref PubMed Scopus (58) Google Scholar, 11Klotz M.G. Schmid M.C. Strous M. op den Camp H.J. Jetten M.S. Hooper A.B. Evolution of an octahaem cytochrome c protein family that is key to aerobic and anaerobic ammonia oxidation by bacteria.Environ. Microbiol. 2008; 10: 3150-3163Crossref PubMed Scopus (117) Google Scholar). HAOs harbor seven bis-His ligated electron-transferring hemes and one 5-coordinate catalytic heme with a His axial ligation (12Igarashi N. Moriyama H. Fujiwara T. Fukumori Y. Tanaka N. The 2.8Å structure of hydroxylamine oxidoreductase from a nitrifying chemoautotrophic bacterium, Nitrosomonas europaea.Nat. Struct. Biol. 1997; 4: 276-284Crossref PubMed Scopus (195) Google Scholar, 13Cedervall P.E. Hooper A.B. Wilmot C.M. Crystallization and preliminary X-ray crystallographic analysis of a new crystal form of hydroxylamine oxidoreductase from Nitrosomonas europaea.Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 2009; 65: 1296-1298Crossref PubMed Scopus (8) Google Scholar, 14Maalcke W.J. Dietl A. Marritt S.J. Butt J.N. Jetten M.S. Keltjens J.T. Barends T.R. Kartal B. Structural basis of biological NO generation by octaheme oxidoreductases.J. Biol. Chem. 2014; 289: 1228-1242Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). In vitro, isolated HAOs are catalytically versatile and can perform hydrazine (N2H4) and hydroxylamine (NH2OH) oxidation, as well as nitrite (NO2−), nitric oxide (NO), and NH2OH reduction, albeit at varying catalytic efficiencies (2Simon J. Kern M. Hermann B. Einsle O. Butt J.N. Physiological function and catalytic versatility of bacterial multihaem cytochromes c involved in nitrogen and sulfur cycling.Biochem. Soc. Trans. 2011; 39: 1864-1870Crossref PubMed Scopus (32) Google Scholar, 15Kostera J. Youngblut M.D. Slosarczyk J.M. Pacheco A.A. Kinetic and product distribution analysis of NO∙ reductase activity in Nitrosomonas europaea hydroxylamine oxidoreductase.J. Biol. Inorg. Chem. 2008; 13: 1073-1083Crossref PubMed Scopus (42) Google Scholar, 16Kostera J. McGarry J. Pacheco A.A. Enzymatic interconversion of ammonia and nitrite: The right tool for the job.Biochemistry. 2010; 49: 8546-8553Crossref PubMed Scopus (31) Google Scholar, 17Hooper A.B. Nason A. Characterization of hydroxylamine-cytochrome c reductase from the chemoautotrophs Nitrosomonas europaea and Nitrosocystis oceanus.J. Biol. Chem. 1965; 240: 4044-4057Abstract Full Text PDF PubMed Google Scholar). Under physiological conditions, however, three of the four structurally characterized representatives perform oxidative catalysis with high catalytic efficiencies (0.1–12 s−1 μM−1) (14Maalcke W.J. Dietl A. Marritt S.J. Butt J.N. Jetten M.S. Keltjens J.T. Barends T.R. Kartal B. Structural basis of biological NO generation by octaheme oxidoreductases.J. Biol. Chem. 2014; 289: 1228-1242Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 18Caranto J.D. Lancaster K.M. Nitric oxide is an obligate bacterial nitrification intermediate produced by hydroxylamine oxidoreductase.Proc. Natl. Acad. Sci. U. S. A. 2017; 114: 8217-8222Crossref PubMed Scopus (179) Google Scholar, 19Maalcke W.J. Reimann J. de Vries S. Butt J.N. Dietl A. Kip N. Mersdorf U. Barends T.R. Jetten M.S. Keltjens J.T. Kartal B. Characterization of anammox hydrazine dehydrogenase, a key N2-producing enzyme in the global nitrogen cycle.J. Biol. Chem. 2016; 291: 17077-17092Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Both the HAO from the aerobic ammonia-oxidizing bacteria—Nitrosomonas europaea (NeHAO) (12Igarashi N. Moriyama H. Fujiwara T. Fukumori Y. Tanaka N. The 2.8Å structure of hydroxylamine oxidoreductase from a nitrifying chemoautotrophic bacterium, Nitrosomonas europaea.Nat. Struct. Biol. 1997; 4: 276-284Crossref PubMed Scopus (195) Google Scholar, 16Kostera J. McGarry J. Pacheco A.A. Enzymatic interconversion of ammonia and nitrite: The right tool for the job.Biochemistry. 2010; 49: 8546-8553Crossref PubMed Scopus (31) Google Scholar, 18Caranto J.D. Lancaster K.M. Nitric oxide is an obligate bacterial nitrification intermediate produced by hydroxylamine oxidoreductase.Proc. Natl. Acad. Sci. U. S. A. 2017; 114: 8217-8222Crossref PubMed Scopus (179) Google Scholar)—and the HAO from the anaerobic ammonium-oxidizing (anammox) bacteria—Kuenenia stuttgartiensis (KsHAO) (14Maalcke W.J. Dietl A. Marritt S.J. Butt J.N. Jetten M.S. Keltjens J.T. Barends T.R. Kartal B. Structural basis of biological NO generation by octaheme oxidoreductases.J. Biol. Chem. 2014; 289: 1228-1242Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar)—catalyze NH2OH oxidation to NO, while hydrazine dehydrogenase from K. stuttgartiensis (KsHDH) oxidizes N2H4 to dinitrogen gas (N2) and is, interestingly, inhibited by both NO and NH2OH at low μM concentrations (19Maalcke W.J. Reimann J. de Vries S. Butt J.N. Dietl A. Kip N. Mersdorf U. Barends T.R. Jetten M.S. Keltjens J.T. Kartal B. Characterization of anammox hydrazine dehydrogenase, a key N2-producing enzyme in the global nitrogen cycle.J. Biol. Chem. 2016; 291: 17077-17092Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 20Akram M. Dietl A. Mersdorf U. Prinz S. Maalcke W. Keltjens J. Ferousi C. de Almeida N.M. Reimann J. Kartal B. Jetten M.S.M. Parey K. Barends T.R.M. A 192-heme electron transfer network in the hydrazine dehydrogenase complex.Sci. Adv. 2019; 5eaav4310Crossref PubMed Scopus (20) Google Scholar). Oxidative HAOs display high tertiary and quaternary similarity, all adopting a homotrimeric configuration (14Maalcke W.J. Dietl A. Marritt S.J. Butt J.N. Jetten M.S. Keltjens J.T. Barends T.R. Kartal B. Structural basis of biological NO generation by octaheme oxidoreductases.J. Biol. Chem. 2014; 289: 1228-1242Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) (or multiples thereof) (20Akram M. Dietl A. Mersdorf U. Prinz S. Maalcke W. Keltjens J. Ferousi C. de Almeida N.M. Reimann J. Kartal B. Jetten M.S.M. Parey K. Barends T.R.M. A 192-heme electron transfer network in the hydrazine dehydrogenase complex.Sci. Adv. 2019; 5eaav4310Crossref PubMed Scopus (20) Google Scholar), with the monomers covalently attached to each other via a unique double cross-link: The C3 and the phenolate oxygen atoms of a Tyr are attached to the C5 and C4 atoms of the catalytic heme moiety of an adjacent subunit. This cross-link results in a diagnostic Soret absorption feature of the reduced protein in the wavelength range of 463 to 475 nm (14Maalcke W.J. Dietl A. Marritt S.J. Butt J.N. Jetten M.S. Keltjens J.T. Barends T.R. Kartal B. Structural basis of biological NO generation by octaheme oxidoreductases.J. Biol. Chem. 2014; 289: 1228-1242Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 19Maalcke W.J. Reimann J. de Vries S. Butt J.N. Dietl A. Kip N. Mersdorf U. Barends T.R. Jetten M.S. Keltjens J.T. Kartal B. Characterization of anammox hydrazine dehydrogenase, a key N2-producing enzyme in the global nitrogen cycle.J. Biol. Chem. 2016; 291: 17077-17092Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 21Andersson K.K. Kent T.A. Lipscomb J.D. Hooper A.B. Münck E. Mössbauer, EPR, and optical studies of the P-460 center of hydroxylamine oxidoreductase from Nitrosomonas.J. Biol. Chem. 1984; 259: 6833-6840Abstract Full Text PDF PubMed Google Scholar), which lends the prosthetic group its name—the P460 cofactor (also see cytochrome P460) (22Caranto J.D. Vilbert A.C. Lancaster K.M. Nitrosomonas europaea cytochrome P460 is a direct link between nitrification and nitrous oxide emission.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: 14704-14709Crossref PubMed Scopus (94) Google Scholar). The Tyr cross-link in the P460 active site of HAO has long been hypothesized to modulate enzyme reactivity toward oxidative catalysis; accordingly, its absence is predicted to favor reduction (11Klotz M.G. Schmid M.C. Strous M. op den Camp H.J. Jetten M.S. Hooper A.B. Evolution of an octahaem cytochrome c protein family that is key to aerobic and anaerobic ammonia oxidation by bacteria.Environ. Microbiol. 2008; 10: 3150-3163Crossref PubMed Scopus (117) Google Scholar, 23Kartal B. de Almeida N.M. Maalcke W.J. Op den Camp H.J. Jetten M.S. Keltjens J.T. How to make a living from anaerobic ammonium oxidation.FEMS Microbiol. Rev. 2013; 37: 428-461Crossref PubMed Scopus (306) Google Scholar). Four HAO genes from εProteobacteria (εHAOs) that lack the critical Tyr residue were recombinantly expressed and shown to reduce NO2− and NH2OH in vitro (Vmax = 1–180 U mg protein−1), while their NH2OH-oxidizing activity was negligible (highest Vmax = 0.07 ± 0.05 U mg protein−1) (24Haase D. Hermann B. Einsle O. Simon J. Epsilonproteobacterial hydroxylamine oxidoreductase (εHao): Characterization of a 'missing link' in the multihaem cytochrome c family.Mol. Microbiol. 2017; 105: 127-138Crossref PubMed Scopus (13) Google Scholar). Additionally, the structure of an HAO—the gene product of kustc0458 from K. stuttgartiensis—that is predicted to be reductive based on physiological experiments (23Kartal B. de Almeida N.M. Maalcke W.J. Op den Camp H.J. Jetten M.S. Keltjens J.T. How to make a living from anaerobic ammonium oxidation.FEMS Microbiol. Rev. 2013; 37: 428-461Crossref PubMed Scopus (306) Google Scholar) and proteomic analyses (25Hu Z. Wessels H. van Alen T. Jetten M.S.M. Kartal B. Nitric oxide-dependent anaerobic ammonium oxidation.Nat. Commun. 2019; 10: 1244Crossref PubMed Scopus (57) Google Scholar), was recently resolved (26Dietl A. Maalcke W.J. Ferousi C. Jetten M.S.M. Kartal B. Barends T.R.M. A 60-heme reductase complex from an anammox bacterium shows an extended electron transfer pathway.Acta Crystallogr. D Struct. Biol. 2019; 75: 333-341Crossref PubMed Scopus (5) Google Scholar). The structure of Kustc0458 revealed the absence of the heme-Tyr cross-link but otherwise highlighted the high structural conservation within the HAO family. Kustc0458 crystallized as a heterododecamer (α6β6) with a diheme cytochrome c (Kustc0457; DH), resulting in a total of 60 hemes for the Kustc0458/7 protein complex (26Dietl A. Maalcke W.J. Ferousi C. Jetten M.S.M. Kartal B. Barends T.R.M. A 60-heme reductase complex from an anammox bacterium shows an extended electron transfer pathway.Acta Crystallogr. D Struct. Biol. 2019; 75: 333-341Crossref PubMed Scopus (5) Google Scholar). Based on the structure, the heterohexamer (α3β3) presents the largest possible heme circuit within the complex and complies with the heme ring arrangement that is seen in all oxidative HAOs. In this case, the heme ring is modified by the addition of two 6-coordinate hemes, contributed by the DH subunits, at each HAO surface heme (heme 1). In analogy with the oxidative HAOs, the Kustc0458/7 catalytic protomer (αβ) comprises nine hemes with saturated coordination—seven HAO bis-His and two DH His/Met ligated hemes—and one 5-coordinate HAO His-ligated heme (heme 4). Kustc0458 is highly transcribed and expressed in K. stuttgartiensis cells under standard laboratory growth conditions (27Kartal B. Maalcke W.J. de Almeida N.M. Cirpus I. Gloerich J. Geerts W. Op den Camp H.J. Harhangi H.R. Janssen-Megens E.M. Francoijs K.J. Stunnenberg H.G. Keltjens J.T. Jetten M.S. Strous M. Molecular mechanism of anaerobic ammonium oxidation.Nature. 2011; 479: 127-130Crossref PubMed Scopus (519) Google Scholar) and has been localized within the anammoxosome (28de Almeida N.M. Neumann S. Mesman R.J. Ferousi C. Keltjens J.T. Jetten M.S. Kartal B. van Niftrik L. Immunogold localization of key metabolic enzymes in the anammoxosome and on the tubule-like structures of Kuenenia stuttgartiensis.J. Bacteriol. 2015; 197: 2432-2441Crossref PubMed Scopus (35) Google Scholar), a bacterial organelle where the main anammox catabolism takes place (29van Niftrik L. Geerts W.J. van Donselaar E.G. Humbel B.M. Yakushevska A. Verkleij A.J. Jetten M.S.M. Strous M. Combined structural and chemical analysis of the anammoxosome: A membrane-bounded intracytoplasmic compartment in anammox bacteria.J. Struct. Biol. 2008; 161: 401-410Crossref PubMed Scopus (146) Google Scholar, 30Neumann S. Wessels H.J. Rijpstra W.I. Sinninghe Damste J.S. Kartal B. Jetten M.S. van Niftrik L. Isolation and characterization of a prokaryotic cell organelle from the anammox bacterium Kuenenia stuttgartiensis.Mol. Microbiol. 2014; 94: 794-802Crossref PubMed Scopus (55) Google Scholar). The anammox pathway proceeds through NO2− reduction to NO, subsequent comproportionation of ammonium (NH4+) and NO by hydrazine synthase (HZS), and the final oxidation of N2H4 to N2 by HDH (Reactions 1–3) (19Maalcke W.J. Reimann J. de Vries S. Butt J.N. Dietl A. Kip N. Mersdorf U. Barends T.R. Jetten M.S. Keltjens J.T. Kartal B. Characterization of anammox hydrazine dehydrogenase, a key N2-producing enzyme in the global nitrogen cycle.J. Biol. Chem. 2016; 291: 17077-17092Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 27Kartal B. Maalcke W.J. de Almeida N.M. Cirpus I. Gloerich J. Geerts W. Op den Camp H.J. Harhangi H.R. Janssen-Megens E.M. Francoijs K.J. Stunnenberg H.G. Keltjens J.T. Jetten M.S. Strous M. Molecular mechanism of anaerobic ammonium oxidation.Nature. 2011; 479: 127-130Crossref PubMed Scopus (519) Google Scholar, 31Dietl A. Ferousi C. Maalcke W.J. Menzel A. de Vries S. Keltjens J.T. Jetten M.S. Kartal B. Barends T.R. The inner workings of the hydrazine synthase multiprotein complex.Nature. 2015; 527: 394-397Crossref PubMed Scopus (73) Google Scholar). The low-potential electrons released from the oxidation of N2H4 are used for cell carbon fixation and other cellular anabolic reactions (23Kartal B. de Almeida N.M. Maalcke W.J. Op den Camp H.J. Jetten M.S. Keltjens J.T. How to make a living from anaerobic ammonium oxidation.FEMS Microbiol. Rev. 2013; 37: 428-461Crossref PubMed Scopus (306) Google Scholar).NO2−+2H++e−→NO+H2O(E0′=+380mV)(1) NO+NH4++2H++3e−→N2H4+H2O(E0′=+60mV)(2) N2H4→N2+4H++4e−(E0′=−750mV)(3) In the absence of nitrite, K. stuttgartiensis can grow on NO and NH4+ instead (25Hu Z. Wessels H. van Alen T. Jetten M.S.M. Kartal B. Nitric oxide-dependent anaerobic ammonium oxidation.Nat. Commun. 2019; 10: 1244Crossref PubMed Scopus (57) Google Scholar). During this NO-dependent anaerobic NH4+ oxidation, transcription of both Kustc0458 and Kustc0457 was strongly downregulated (28- and 39-fold, respectively). Additionally, the only NO-forming nitrite reductase identified in the K. stuttgartiensis genome (cd1NiR homolog) had low abundance in both transcriptomes and proteomes obtained under standard and NO-dependent growth conditions (25Hu Z. Wessels H. van Alen T. Jetten M.S.M. Kartal B. Nitric oxide-dependent anaerobic ammonium oxidation.Nat. Commun. 2019; 10: 1244Crossref PubMed Scopus (57) Google Scholar, 27Kartal B. Maalcke W.J. de Almeida N.M. Cirpus I. Gloerich J. Geerts W. Op den Camp H.J. Harhangi H.R. Janssen-Megens E.M. Francoijs K.J. Stunnenberg H.G. Keltjens J.T. Jetten M.S. Strous M. Molecular mechanism of anaerobic ammonium oxidation.Nature. 2011; 479: 127-130Crossref PubMed Scopus (519) Google Scholar). Furthermore, neither cd1 cytochrome (cd1NiR) nor copper-containing (CuNiR) nitrite reductase homologs are conserved among anammox genomes, while the orthologs of Kustc0458 are present in all known anammox genera (26Dietl A. Maalcke W.J. Ferousi C. Jetten M.S.M. Kartal B. Barends T.R.M. A 60-heme reductase complex from an anammox bacterium shows an extended electron transfer pathway.Acta Crystallogr. D Struct. Biol. 2019; 75: 333-341Crossref PubMed Scopus (5) Google Scholar). Based on in vivo observations from K. stuttgartiensis, Kustc0458 was implicated in the reduction of nitrite to NO, the first step of the anammox pathway (23Kartal B. de Almeida N.M. Maalcke W.J. Op den Camp H.J. Jetten M.S. Keltjens J.T. How to make a living from anaerobic ammonium oxidation.FEMS Microbiol. Rev. 2013; 37: 428-461Crossref PubMed Scopus (306) Google Scholar). In this study, we purified the native Kustc0458/7 complex from K. stuttgartiensis biomass, assessed its catalytic potential involving physiologically relevant nitrogen species and showed that this protein reduces nitrite to NO. Our findings are examined in the context of the HAO protein family, and the physiological relevance of Kustc0458/7 is discussed. Kustc0458 was consistently purified from K. stuttgartiensis single-cell enrichment cultures in an equimolar complex with Kustc0457. Native and sodium dodecyl sulfate (SDS)-denaturing polyacrylamide gel electrophoresis (PAGE) migration profiles indicated a stable, noncovalent dodecameric composition for the complex in solution that is in agreement with the crystallographically resolved structure (26Dietl A. Maalcke W.J. Ferousi C. Jetten M.S.M. Kartal B. Barends T.R.M. A 60-heme reductase complex from an anammox bacterium shows an extended electron transfer pathway.Acta Crystallogr. D Struct. Biol. 2019; 75: 333-341Crossref PubMed Scopus (5) Google Scholar). The electronic absorption spectra of the fully reduced protein revealed the absence of the characteristic P460 absorption feature (Fig. 1), in line with the absence of the Tyr cross-link. Fully oxidized (as isolated) Kustc0458/7 displayed a Soret maximum at 409 nm, which broadened and shifted to 415 nm upon partial reduction by ascorbate. Full reduction by dithionite resulted in a sharp Soret at 419.5 nm, a subtle shoulder at 425 nm, and Q band maxima at 524 and 553 nm. Notably, despite the Met axial ligation of both DH heme iron centers (26Dietl A. Maalcke W.J. Ferousi C. Jetten M.S.M. Kartal B. Barends T.R.M. A 60-heme reductase complex from an anammox bacterium shows an extended electron transfer pathway.Acta Crystallogr. D Struct. Biol. 2019; 75: 333-341Crossref PubMed Scopus (5) Google Scholar), no charge transfer band at 695 nm was observed. To test whether the Tyr cross-link is necessary for oxidative catalysis, we probed the capacity of Kustc0458/7 to oxidize NH2OH and N2H4 spectroscopically. Depending on the sample preparation, the measured rates were ranging from zero to about 0.020 U mg−1 for both substrates, which is 100- to 1000-fold slower compared with the anammox oxidative HAOs and NeHAO, respectively. Notably, both anammox oxidative HAOs are among the most abundant proteins in K. stuttgartiensis cells (27Kartal B. Maalcke W.J. de Almeida N.M. Cirpus I. Gloerich J. Geerts W. Op den Camp H.J. Harhangi H.R. Janssen-Megens E.M. Francoijs K.J. Stunnenberg H.G. Keltjens J.T. Jetten M.S. Strous M. Molecular mechanism of anaerobic ammonium oxidation.Nature. 2011; 479: 127-130Crossref PubMed Scopus (519) Google Scholar) and hence, even a minute (i.e., ∼0.5%) contamination of the Kustc0458/7 sample with either KsHAO or KsHDH (for NH2OH or N2H4 oxidation, respectively) could explain the measured rates, strongly suggesting that Kustc0458/7 is not an oxidative catalyst. Reductive reactivity of Kustc0458/7 toward NO2−, NO, and NH2OH was investigated by monitoring the oxidation of the methyl viologen monocation radical (MVred) (32Lawton T.J. Bowen K.E. Sayavedra-Soto L.A. Arp D.J. Rosenzweig A.C. Characterization of a nitrite reductase involved in nitrifier denitrification.J. Biol. Chem. 2013; 288: 25575-25583Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). At 50 μM substrate concentration, NO2− reduction proceeded at 0.1 U mg−1 while NO and NH2OH were consistently faster (2.8 and 4.6 U mg−1, respectively). Similar kinetic trends have been observed for the ammonia-producing respiratory cytochrome c nitrite reductase (ccNiR) and were explained by the presumed higher activation entropy for hydroxylamine reduction (33van Wonderen J.H. Burlat B. Richardson D.J. Cheesman M.R. Butt J.N. The nitric oxide reductase activity of cytochrome c nitrite reductase from Escherichia coli.J. Biol. Chem. 2008; 283: 9587-9594Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Colorimetric determination of ammonia after completion of each reductive assay routinely returned the same substrate-to-product ratios: 1:0.6, 1:1, and 1:1 for NO2−, NO, and NH2OH, respectively. The stoichiometric nitrogen imbalance observed for nitrite conversion is presumably due to the release of at least one catalytic intermediate from the Kustc0458/7 active site during catalysis in vitro. Given the reducing environment in the assay achieved by excess MVred and the higher catalytic rates of Kustc0458/7 for the downstream reactions at relevant concentrations, i.e., NO and NH2OH reduction, this observation could only be attributed to finely tuned binding equilibria that could possibly be affected by redox modulations of the extended 9-heme network per active site. In order to determine whether Kustc0458/7 performed its predicted physiological function of nitrite reduction to NO, different artificial electron carriers were used with nitrite as the substrate. The reducing environment created by excess MVred and the reactivity of NO prevented the direct measurement of NO when MVred was used as the electron carrier. To circumvent this problem, phenazine ethosulfate, which has a much higher midpoint potential (E′0 = +55 mV) compared with MV (E′0 = −440 mV), was used as an electron carrier to directly measure NO production in membrane-inlet mass spectrometry (MIMS) experiments. Here, when the reaction was initiated with the addition of nitrite, stable and reproducible NO production was detected at a rate of 0.52 mU mg−1 (Fig. 2). Colorimetric nitrite measurements at the end of these experiments showed that all nitrite reduction could be accounted for by the produced NO and no other nitrogenous species such as hydroxylamine or ammonium were produced. Based on these results, we conclude that the Kustc0458/7 protein complex is a NO-producing reductive HAO that is incompetent for oxidative catalysis. Whereas HAOr from K. stuttgartiensis and HAOs from Epsilonproteobacteria all lack appreciable in vitro oxidative activity (24Haase D. Hermann B. Einsle O. Simon J. Epsilonproteobacterial hydroxylamine oxidoreductase (εHao): Characterization of a 'missing link' in the multihaem cytochrome c family.Mol. Microbiol. 2017; 105: 127-138Crossref PubMed Scopus (13) Google Scholar), oxidative HAO complexes, conversely, have been shown to act as reductases in vitro, albeit with diverse rates. Both NeHAO and KsHAO can reduce NO2−, and NeHAO can also reduce NO and NH2OH, demonstrating that the HAO active site architecture is inherently competent for substrate reduction (14Maalcke W.J. Dietl A. Marritt S.J. Butt J.N. Jetten M.S. Keltjens J.T. Barends T.R. Kartal B. Structural basis of biological NO generation by octaheme oxidoreductases.J. Biol. Chem. 2014; 289: 1228-1242Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 15Kostera J. Youngblut M.D. Slosarczyk J.M. Pacheco A.A. Kinetic and product distribution analysis of NO∙ reductase activity in Nitrosomo

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