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Peter M H Kroneck - One of the best experts on this subject based on the ideXlab platform.
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a napc nirt type cytochrome c nrfh is the mediator between the quinone pool and the cytochrome c Nitrite Reductase of wolinella succinogenes
Molecular Microbiology, 2002Co-Authors: Jörg Simon, Achim Kroger, Oliver Einsle, Peter M H Kroneck, Roland Gross, Oliver KlimmekAbstract:: Wolinella succinogenes can grow by anaerobic respiration with nitrate or Nitrite using formate as electron donor. Two forms of Nitrite Reductase were isolated from the membrane fraction of W. succinogenes. One form consisted of a 58 kDa polypeptide (NrfA) that was identical to the periplasmic Nitrite Reductase. The other form consisted of NrfA and a 22 kDa polypeptide (NrfH). Both forms catalysed Nitrite reduction by reduced benzyl viologen, but only the dimeric form catalysed Nitrite reduction by dimethylnaphthoquinol. Liposomes containing heterodimeric Nitrite Reductase, formate dehydrogenase and menaquinone catalysed the electron transport from formate to Nitrite; this was coupled to the generation of an electrochemical proton potential (positive outside) across the liposomal membrane. It is concluded that the electron transfer from menaquinol to the catalytic subunit (NrfA) of W. succinogenes Nitrite Reductase is mediated by NrfH. The structural genes nrfA and nrfH were identified in an apparent operon (nrfHAIJ) with two additional genes. The gene nrfA encodes the precursor of NrfA carrying an N-terminal signal peptide (22 residues). NrfA (485 residues) is predicted to be a hydrophilic protein that is similar to the NrfA proteins of Sulfurospirillum deleyianum and of Escherichia coli. NrfH (177 residues) is predicted to be a membrane-bound tetrahaem cytochrome c belonging to the NapC/NirT family. The products of nrfI and nrfJ resemble proteins involved in cytochrome c biogenesis. The C-terminal third of NrfI (902 amino acid residues) is similar to CcsA proteins from Gram-positive bacteria, cyanobacteria and chloroplasts. The residual N-terminal part of NrfI resembles Ccs1 proteins. The deduced NrfJ protein resembles the thioredoxin-like proteins (ResA) of Helicobacter pylori and of Bacillus subtilis, but lacks the common motif CxxC of ResA. The properties of three deletion mutants of W. succinogenes (DeltanrfJ, DeltanrfIJ and DeltanrfAIJ) were studied. Mutants DeltanrfAIJ and DeltanrfIJ did not grow with Nitrite as terminal electron acceptor or with nitrate in the absence of NH4+ and lacked Nitrite Reductase activity, whereas mutant DeltanrfJ showed wild-type properties. The NrfA protein formed by mutant DeltanrfIJ seemed to lack part of the haem C, suggesting that NrfI is involved in NrfA maturation.
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crystallization and preliminary x ray analysis of the membrane bound cytochrome c Nitrite Reductase complex nrfha from wolinella succinogenes
Acta Crystallographica Section D-biological Crystallography, 2002Co-Authors: Oliver Einsle, Jörg Simon, Oliver Klimmek, Petra Stach, Albrecht Messerschmidt, Achim Kroger, Peter M H KroneckAbstract:Crystals of the complex between the enzyme cytochrome c Nitrite Reductase (NrfA) and the membrane-bound quinol oxidase and electron carrier NrfH were grown by vapour diffusion using ammonium sulfate as a precipitant. In the ∊-proteobacterium Wolinella succinogenes, NrfA and NrfH form a functional membrane-bound complex which catalyzes the last step in the metabolic pathway of nitrate dissimilation. NrfH represents a prototype of a large family of putative bacterial quinol oxidases, the NapC/NirT family, which have been proposed to serve as electron donors for a variety of Reductases. Crystal growth of the NrfHA complex was strongly dependent on the presence of detergent; the crystals grown belonged to space group I422.
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pentahaem cytochrome c Nitrite Reductase reaction with hydroxylamine a potential reaction intermediate and substrate
Biochemical Society Transactions, 2001Co-Authors: Marc Rudolf, Oliver Einsle, Frank Neese, Peter M H KroneckAbstract:The pentahaem enzyme cytochrome c Nitrite Reductase catalyses the reduction of Nitrite to ammonia, a key reaction in the biological nitrogen cycle. The enzyme can also transform nitrogen monoxide and hydroxylamine, two potential bound reaction intermediates, into ammonia. Structural and mechanistic aspects of the multihaem enzyme are discussed in comparison with hydroxylamine oxidoReductase, a trimeric protein with eight haem molecules per subunit.
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cytochrome c Nitrite Reductase from wolinella succinogenes structure at 1 6 a resolution inhibitor binding and heme packing motifs
Journal of Biological Chemistry, 2000Co-Authors: Oliver Einsle, Jörg Simon, Petra Stach, Albrecht Messerschmidt, Achim Kroger, Robert Huber, Peter M H KroneckAbstract:Cytochrome c Nitrite Reductase catalyzes the 6-electron reduction of Nitrite to ammonia. This second part of the respiratory pathway of nitrate ammonification is a key step in the biological nitrogen cycle. The x-ray structure of the enzyme from the epsilon-proteobacterium Wolinella succinogenes has been solved to a resolution of 1.6 A. It is a pentaheme c-type cytochrome whose heme groups are packed in characteristic motifs that also occur in other multiheme cytochromes. Structures of W. succinogenes Nitrite Reductase have been obtained with water bound to the active site heme iron as well as complexes with two inhibitors, sulfate and azide, whose binding modes and inhibitory functions differ significantly. Cytochrome c Nitrite Reductase is part of a highly optimized respiratory system found in a wide range of Gram-negative bacteria. It reduces both anionic and neutral substrates at the distal side of a lysine-coordinated high-spin heme group, which is accessible through two different channels, allowing for a guided flow of reaction educt and product. Based on sequence comparison and secondary structure prediction, we have demonstrated that cytochrome c Nitrite Reductases constitute a protein family of high structural similarity.
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bacterial cytochrome c Nitrite Reductase new structural and functional aspects
Journal of Inorganic Biochemistry, 2000Co-Authors: Petra Stach, Oliver Einsle, Wolfram Schumacher, E Kurun, Peter M H KroneckAbstract:Cytochrome c Nitrite Reductase catalyzes the six-electron reduction of Nitrite to ammonia as a key step within the biological nitrogen cycle. Most recently, the crystal structure of the soluble enzyme from Sulfurospirillum deleyianum could be solved to 1.9 A resolution. This set the basis for new experiments on structural and functional aspects of the pentaheme protein which carries a Ca(2+) ion close to the active site heme. In the crystal, the protein was a homodimer with ten hemes in very close packing. The strong interaction between the Nitrite Reductase monomers also occurred in solution according to the dependence of the activity on the protein concentration. Addition of Ca(2+) to the enzyme as isolated had a stimulating effect on the activity. Ca(2+) could be removed from the enzyme by treatment with chelating agents such as EGTA or EDTA which led to a decrease in activity. In addition to Nitrite, the enzyme converted NO, hydroxylamine and O-methyl hydroxylamine to ammonia at considerable rates. With N2O the activity was much lower; most likely dinitrogen was the product in this case. Cytochrome c Nitrite Reductase exhibited a remarkably high sulfite Reductase activity, with hydrogen sulfide as the product. A paramagnetic Fe(II)-NO, S = 1/2 adduct was identified by rapid freeze EPR spectroscopy under turnover conditions with Nitrite. This potential reaction intermediate of the reduction of Nitrite to ammonia was also observed with PAPA NONOate and Spermine NONOate.
Oliver Einsle - One of the best experts on this subject based on the ideXlab platform.
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a napc nirt type cytochrome c nrfh is the mediator between the quinone pool and the cytochrome c Nitrite Reductase of wolinella succinogenes
Molecular Microbiology, 2002Co-Authors: Jörg Simon, Achim Kroger, Oliver Einsle, Peter M H Kroneck, Roland Gross, Oliver KlimmekAbstract:: Wolinella succinogenes can grow by anaerobic respiration with nitrate or Nitrite using formate as electron donor. Two forms of Nitrite Reductase were isolated from the membrane fraction of W. succinogenes. One form consisted of a 58 kDa polypeptide (NrfA) that was identical to the periplasmic Nitrite Reductase. The other form consisted of NrfA and a 22 kDa polypeptide (NrfH). Both forms catalysed Nitrite reduction by reduced benzyl viologen, but only the dimeric form catalysed Nitrite reduction by dimethylnaphthoquinol. Liposomes containing heterodimeric Nitrite Reductase, formate dehydrogenase and menaquinone catalysed the electron transport from formate to Nitrite; this was coupled to the generation of an electrochemical proton potential (positive outside) across the liposomal membrane. It is concluded that the electron transfer from menaquinol to the catalytic subunit (NrfA) of W. succinogenes Nitrite Reductase is mediated by NrfH. The structural genes nrfA and nrfH were identified in an apparent operon (nrfHAIJ) with two additional genes. The gene nrfA encodes the precursor of NrfA carrying an N-terminal signal peptide (22 residues). NrfA (485 residues) is predicted to be a hydrophilic protein that is similar to the NrfA proteins of Sulfurospirillum deleyianum and of Escherichia coli. NrfH (177 residues) is predicted to be a membrane-bound tetrahaem cytochrome c belonging to the NapC/NirT family. The products of nrfI and nrfJ resemble proteins involved in cytochrome c biogenesis. The C-terminal third of NrfI (902 amino acid residues) is similar to CcsA proteins from Gram-positive bacteria, cyanobacteria and chloroplasts. The residual N-terminal part of NrfI resembles Ccs1 proteins. The deduced NrfJ protein resembles the thioredoxin-like proteins (ResA) of Helicobacter pylori and of Bacillus subtilis, but lacks the common motif CxxC of ResA. The properties of three deletion mutants of W. succinogenes (DeltanrfJ, DeltanrfIJ and DeltanrfAIJ) were studied. Mutants DeltanrfAIJ and DeltanrfIJ did not grow with Nitrite as terminal electron acceptor or with nitrate in the absence of NH4+ and lacked Nitrite Reductase activity, whereas mutant DeltanrfJ showed wild-type properties. The NrfA protein formed by mutant DeltanrfIJ seemed to lack part of the haem C, suggesting that NrfI is involved in NrfA maturation.
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crystallization and preliminary x ray analysis of the membrane bound cytochrome c Nitrite Reductase complex nrfha from wolinella succinogenes
Acta Crystallographica Section D-biological Crystallography, 2002Co-Authors: Oliver Einsle, Jörg Simon, Oliver Klimmek, Petra Stach, Albrecht Messerschmidt, Achim Kroger, Peter M H KroneckAbstract:Crystals of the complex between the enzyme cytochrome c Nitrite Reductase (NrfA) and the membrane-bound quinol oxidase and electron carrier NrfH were grown by vapour diffusion using ammonium sulfate as a precipitant. In the ∊-proteobacterium Wolinella succinogenes, NrfA and NrfH form a functional membrane-bound complex which catalyzes the last step in the metabolic pathway of nitrate dissimilation. NrfH represents a prototype of a large family of putative bacterial quinol oxidases, the NapC/NirT family, which have been proposed to serve as electron donors for a variety of Reductases. Crystal growth of the NrfHA complex was strongly dependent on the presence of detergent; the crystals grown belonged to space group I422.
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pentahaem cytochrome c Nitrite Reductase reaction with hydroxylamine a potential reaction intermediate and substrate
Biochemical Society Transactions, 2001Co-Authors: Marc Rudolf, Oliver Einsle, Frank Neese, Peter M H KroneckAbstract:The pentahaem enzyme cytochrome c Nitrite Reductase catalyses the reduction of Nitrite to ammonia, a key reaction in the biological nitrogen cycle. The enzyme can also transform nitrogen monoxide and hydroxylamine, two potential bound reaction intermediates, into ammonia. Structural and mechanistic aspects of the multihaem enzyme are discussed in comparison with hydroxylamine oxidoReductase, a trimeric protein with eight haem molecules per subunit.
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cytochrome c Nitrite Reductase from wolinella succinogenes structure at 1 6 a resolution inhibitor binding and heme packing motifs
Journal of Biological Chemistry, 2000Co-Authors: Oliver Einsle, Jörg Simon, Petra Stach, Albrecht Messerschmidt, Achim Kroger, Robert Huber, Peter M H KroneckAbstract:Cytochrome c Nitrite Reductase catalyzes the 6-electron reduction of Nitrite to ammonia. This second part of the respiratory pathway of nitrate ammonification is a key step in the biological nitrogen cycle. The x-ray structure of the enzyme from the epsilon-proteobacterium Wolinella succinogenes has been solved to a resolution of 1.6 A. It is a pentaheme c-type cytochrome whose heme groups are packed in characteristic motifs that also occur in other multiheme cytochromes. Structures of W. succinogenes Nitrite Reductase have been obtained with water bound to the active site heme iron as well as complexes with two inhibitors, sulfate and azide, whose binding modes and inhibitory functions differ significantly. Cytochrome c Nitrite Reductase is part of a highly optimized respiratory system found in a wide range of Gram-negative bacteria. It reduces both anionic and neutral substrates at the distal side of a lysine-coordinated high-spin heme group, which is accessible through two different channels, allowing for a guided flow of reaction educt and product. Based on sequence comparison and secondary structure prediction, we have demonstrated that cytochrome c Nitrite Reductases constitute a protein family of high structural similarity.
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bacterial cytochrome c Nitrite Reductase new structural and functional aspects
Journal of Inorganic Biochemistry, 2000Co-Authors: Petra Stach, Oliver Einsle, Wolfram Schumacher, E Kurun, Peter M H KroneckAbstract:Cytochrome c Nitrite Reductase catalyzes the six-electron reduction of Nitrite to ammonia as a key step within the biological nitrogen cycle. Most recently, the crystal structure of the soluble enzyme from Sulfurospirillum deleyianum could be solved to 1.9 A resolution. This set the basis for new experiments on structural and functional aspects of the pentaheme protein which carries a Ca(2+) ion close to the active site heme. In the crystal, the protein was a homodimer with ten hemes in very close packing. The strong interaction between the Nitrite Reductase monomers also occurred in solution according to the dependence of the activity on the protein concentration. Addition of Ca(2+) to the enzyme as isolated had a stimulating effect on the activity. Ca(2+) could be removed from the enzyme by treatment with chelating agents such as EGTA or EDTA which led to a decrease in activity. In addition to Nitrite, the enzyme converted NO, hydroxylamine and O-methyl hydroxylamine to ammonia at considerable rates. With N2O the activity was much lower; most likely dinitrogen was the product in this case. Cytochrome c Nitrite Reductase exhibited a remarkably high sulfite Reductase activity, with hydrogen sulfide as the product. A paramagnetic Fe(II)-NO, S = 1/2 adduct was identified by rapid freeze EPR spectroscopy under turnover conditions with Nitrite. This potential reaction intermediate of the reduction of Nitrite to ammonia was also observed with PAPA NONOate and Spermine NONOate.
S S Hasnain - One of the best experts on this subject based on the ideXlab platform.
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unexpected roles of a tether harboring a tyrosine gatekeeper residue in modular Nitrite Reductase catalysis
ACS Catalysis, 2019Co-Authors: Tobias M Hedison, Derren J Heyes, Andreea I Iorgu, Robert R Eady, Rajesh T Shenoy, Karl Fisher, Gareth S A Wright, S V Antonyuk, S S Hasnain, Nigel S ScruttonAbstract:It is generally assumed that tethering enhances rates of electron harvesting and delivery to active sites in multidomain enzymes by proximity and sampling mechanisms. Here, we explore this idea in a tethered 3-domain, trimeric copper-containing Nitrite Reductase. By reverse engineering, we find that tethering does not enhance the rate of electron delivery from its pendant cytochrome c to the catalytic copper-containing core. Using a linker that harbors a gatekeeper tyrosine in a Nitrite access channel, the tethered haem domain enables catalysis by other mechanisms. Tethering communicates the redox state of the haem to the distant T2Cu center that helps initiate substrate binding for catalysis. It also tunes copper reduction potentials, suppresses reductive enzyme inactivation, enhances enzyme affinity for substrate, and promotes intercopper electron transfer. Tethering has multiple unanticipated beneficial roles, the combination of which fine-tunes function beyond simplistic mechanisms expected from proxi...
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unexpected roles of a tether harboring a tyrosine gatekeeper residue in modular Nitrite Reductase catalysis
ACS Catalysis, 2019Co-Authors: Tobias M Hedison, Derren J Heyes, Andreea I Iorgu, Robert R Eady, Rajesh T Shenoy, Karl Fisher, Gareth S A Wright, S V Antonyuk, Sam Hay, S S HasnainAbstract:© 2019 American Chemical Society. It is generally assumed that tethering enhances rates of electron harvesting and delivery to active sites in multidomain enzymes by proximity and sampling mechanisms. Here, we explore this idea in a tethered 3-domain, trimeric copper-containing Nitrite Reductase. By reverse engineering, we find that tethering does not enhance the rate of electron delivery from its pendant cytochrome c to the catalytic copper-containing core. Using a linker that harbors a gatekeeper tyrosine in a Nitrite access channel, the tethered haem domain enables catalysis by other mechanisms. Tethering communicates the redox state of the haem to the distant T2Cu center that helps initiate substrate binding for catalysis. It also tunes copper reduction potentials, suppresses reductive enzyme inactivation, enhances enzyme affinity for substrate, and promotes intercopper electron transfer. Tethering has multiple unanticipated beneficial roles, the combination of which fine-tunes function beyond simplistic mechanisms expected from proximity and restrictive sampling models.
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proton coupled electron transfer in the catalytic cycle of alcaligenes xylosoxidans copper dependent Nitrite Reductase
Biochemistry, 2011Co-Authors: Nicole G H Leferink, Derren J Heyes, Nigel S Scrutton, Robert R Eady, S V Antonyuk, Stephen E J Rigby, M A Hough, S S HasnainAbstract:We demonstrated recently that two protons are involved in reduction of Nitrite to nitric oxide through a proton-coupled electron transfer (ET) reaction catalyzed by the blue Cu-dependent Nitrite Reductase (Cu NiR) of Alcaligenes xylosoxidans (AxNiR). Here, the functionality of two putative proton channels, one involving Asn90 and the other His254, is studied using single (N90S, H254F) and double (N90S–H254F) mutants. All mutants studied are active, indicating that protons are still able to reach the active site. The H254F mutation has no effect on the catalytic activity, while the N90S mutation results in ∼70% decrease in activity. Laser flash-photolysis experiments show that in H254F and wild-type enzyme electrons enter at the level of the T1Cu and then redistribute between the two Cu sites. Complete ET from T1Cu to T2Cu occurs only when Nitrite binds at the T2Cu site. This indicates that substrate binding to T2Cu promotes ET from T1Cu, suggesting that the enzyme operates an ordered mechanism. In fact, i...
Tobias M Hedison - One of the best experts on this subject based on the ideXlab platform.
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unexpected roles of a tether harboring a tyrosine gatekeeper residue in modular Nitrite Reductase catalysis
ACS Catalysis, 2019Co-Authors: Tobias M Hedison, Derren J Heyes, Andreea I Iorgu, Robert R Eady, Rajesh T Shenoy, Karl Fisher, Gareth S A Wright, S V Antonyuk, Sam Hay, S S HasnainAbstract:© 2019 American Chemical Society. It is generally assumed that tethering enhances rates of electron harvesting and delivery to active sites in multidomain enzymes by proximity and sampling mechanisms. Here, we explore this idea in a tethered 3-domain, trimeric copper-containing Nitrite Reductase. By reverse engineering, we find that tethering does not enhance the rate of electron delivery from its pendant cytochrome c to the catalytic copper-containing core. Using a linker that harbors a gatekeeper tyrosine in a Nitrite access channel, the tethered haem domain enables catalysis by other mechanisms. Tethering communicates the redox state of the haem to the distant T2Cu center that helps initiate substrate binding for catalysis. It also tunes copper reduction potentials, suppresses reductive enzyme inactivation, enhances enzyme affinity for substrate, and promotes intercopper electron transfer. Tethering has multiple unanticipated beneficial roles, the combination of which fine-tunes function beyond simplistic mechanisms expected from proximity and restrictive sampling models.
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unexpected roles of a tether harboring a tyrosine gatekeeper residue in modular Nitrite Reductase catalysis
ACS Catalysis, 2019Co-Authors: Tobias M Hedison, Derren J Heyes, Andreea I Iorgu, Robert R Eady, Rajesh T Shenoy, Karl Fisher, Gareth S A Wright, S V Antonyuk, S S Hasnain, Nigel S ScruttonAbstract:It is generally assumed that tethering enhances rates of electron harvesting and delivery to active sites in multidomain enzymes by proximity and sampling mechanisms. Here, we explore this idea in a tethered 3-domain, trimeric copper-containing Nitrite Reductase. By reverse engineering, we find that tethering does not enhance the rate of electron delivery from its pendant cytochrome c to the catalytic copper-containing core. Using a linker that harbors a gatekeeper tyrosine in a Nitrite access channel, the tethered haem domain enables catalysis by other mechanisms. Tethering communicates the redox state of the haem to the distant T2Cu center that helps initiate substrate binding for catalysis. It also tunes copper reduction potentials, suppresses reductive enzyme inactivation, enhances enzyme affinity for substrate, and promotes intercopper electron transfer. Tethering has multiple unanticipated beneficial roles, the combination of which fine-tunes function beyond simplistic mechanisms expected from proxi...
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solvent slaved protein motions accompany proton coupled electron transfer reactions catalysed by copper Nitrite Reductase
Chemical Communications, 2019Co-Authors: Tobias M Hedison, Derren J Heyes, Muralidharan Shanmugam, Andreea I Iorgu, Nigel S ScruttonAbstract:Through the use of time-resolved pH-jump spectroscopy, we demonstrate how proton transfer is coupled to inter-copper electron transfer in a copper Nitrite Reductase (CuNiR). Combined use of electron paramagnetic resonance spectroscopy with solvent viscosity- and pressure-dependence pH-jump stopped-flow spectroscopy is used to show that solvent-slaved protein motions are linked to this proton coupled electron transfer step in CuNiR.
Nigel S Scrutton - One of the best experts on this subject based on the ideXlab platform.
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unexpected roles of a tether harboring a tyrosine gatekeeper residue in modular Nitrite Reductase catalysis
ACS Catalysis, 2019Co-Authors: Tobias M Hedison, Derren J Heyes, Andreea I Iorgu, Robert R Eady, Rajesh T Shenoy, Karl Fisher, Gareth S A Wright, S V Antonyuk, S S Hasnain, Nigel S ScruttonAbstract:It is generally assumed that tethering enhances rates of electron harvesting and delivery to active sites in multidomain enzymes by proximity and sampling mechanisms. Here, we explore this idea in a tethered 3-domain, trimeric copper-containing Nitrite Reductase. By reverse engineering, we find that tethering does not enhance the rate of electron delivery from its pendant cytochrome c to the catalytic copper-containing core. Using a linker that harbors a gatekeeper tyrosine in a Nitrite access channel, the tethered haem domain enables catalysis by other mechanisms. Tethering communicates the redox state of the haem to the distant T2Cu center that helps initiate substrate binding for catalysis. It also tunes copper reduction potentials, suppresses reductive enzyme inactivation, enhances enzyme affinity for substrate, and promotes intercopper electron transfer. Tethering has multiple unanticipated beneficial roles, the combination of which fine-tunes function beyond simplistic mechanisms expected from proxi...
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solvent slaved protein motions accompany proton coupled electron transfer reactions catalysed by copper Nitrite Reductase
Chemical Communications, 2019Co-Authors: Tobias M Hedison, Derren J Heyes, Muralidharan Shanmugam, Andreea I Iorgu, Nigel S ScruttonAbstract:Through the use of time-resolved pH-jump spectroscopy, we demonstrate how proton transfer is coupled to inter-copper electron transfer in a copper Nitrite Reductase (CuNiR). Combined use of electron paramagnetic resonance spectroscopy with solvent viscosity- and pressure-dependence pH-jump stopped-flow spectroscopy is used to show that solvent-slaved protein motions are linked to this proton coupled electron transfer step in CuNiR.
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proton coupled electron transfer in the catalytic cycle of alcaligenes xylosoxidans copper dependent Nitrite Reductase
Biochemistry, 2011Co-Authors: Nicole G H Leferink, Derren J Heyes, Nigel S Scrutton, Robert R Eady, S V Antonyuk, Stephen E J Rigby, M A Hough, S S HasnainAbstract:We demonstrated recently that two protons are involved in reduction of Nitrite to nitric oxide through a proton-coupled electron transfer (ET) reaction catalyzed by the blue Cu-dependent Nitrite Reductase (Cu NiR) of Alcaligenes xylosoxidans (AxNiR). Here, the functionality of two putative proton channels, one involving Asn90 and the other His254, is studied using single (N90S, H254F) and double (N90S–H254F) mutants. All mutants studied are active, indicating that protons are still able to reach the active site. The H254F mutation has no effect on the catalytic activity, while the N90S mutation results in ∼70% decrease in activity. Laser flash-photolysis experiments show that in H254F and wild-type enzyme electrons enter at the level of the T1Cu and then redistribute between the two Cu sites. Complete ET from T1Cu to T2Cu occurs only when Nitrite binds at the T2Cu site. This indicates that substrate binding to T2Cu promotes ET from T1Cu, suggesting that the enzyme operates an ordered mechanism. In fact, i...