Probing the Early Steps in the Catalytic Reduction of Nitrite to Ammonia, Catalyzed By Cytochrome C Nitrite Reductase (original) (raw)

Cytochrome c Nitrite Reductase (ccNiR) is a periplasmic, decaheme homodimeric enzyme that catalyzes the six-electron reduction of nitrite to ammonia. Under standard assay conditions catalysis proceeds without detected intermediates, and it's been assumed that this is also true in vivo. However, this report demonstrates that in vitro it's possible to trap putative intermediates by controlling the electrochemical potential at which reduction takes place. Such experiments provide valuable insights regarding ccNiR-catalyzed nitrite ammonification. UV/Vis spectropotentiometry showed that nitrite-loaded Shewanella oneidensis ccNiR is reduced in a concerted 2-electron step to generate an {FeNO} 7 moiety at the active site, with an associated midpoint potential of +246 mV vs SHE at pH 7. By contrast, cyanide-bound active site reduction is a one-electron process with a midpoint potential of 20 mV, and without a strong-field ligand the active site midpoint potential shifts 70 mV lower still. EPR analysis subsequently revealed that the {FeNO} 7 moiety possesses an unusual spectral signature, different from those normally observed for {FeNO} 7 hemes, that may indicate magnetic interaction of the active site with nearby hemes. Protein film voltammetry experiments previously showed that catalytic nitrite reduction to ammonia by S. oneidensis ccNiR requires an applied potential of at least −120 mV, well below iii the midpoint potential for {FeNO} 7 formation. Thus, it appears that an {FeNO} 7 active site is a catalytic intermediate in the ccNiR-mediated reduction of nitrite to ammonia, whose degree of accumulation depends exclusively on the applied potential. At low potentials the species is rapidly reduced and doesn't accumulate, while at higher potentials it is trapped, thus preventing catalytic ammonia formation. When the weak reductant ferrocyanide is used as the electron source, S. oneidensis ccNiR catalyzes the one-electron reduction of nitrite to nitric oxide. The reaction rate has hyperbolic dependence on nitrite concentration and linear dependence on ccNiR concentration. NO⋅ release is minimal compared to the rate of ammonia formation at lower applied potentials. Kinetic studies also show that the rate of NO⋅ production is pH-dependent, and that an amino acid with pKa of 6.9, probably His268, needs to be protonated for the enzyme to be active. iv TABLE OF CONTENTS CHAPTERS PAGE NUMBER xv LIST OF SCHEMES Scheme 1.1. The half reaction for nitrogen fixation. The overall reaction is catalyzed by nitrogenase (NiF in Fig. 1.1)…………………………………………..………………………3 Scheme 1.2. The half reactions for nitrification. The first step is catalyzed by the enzyme ammonia monooxygenase (AMO, Fig. 1.1), the second by hydroxylamine oxidase (HAO, Fig. 1.1), and the third by nitrite oxidoreductase (NXR, Fig. 1.1). 3 The first two steps are carried out by ammonia oxidizing bacteria and archaea (AOB, AOA), the final one by nitrite oxidizers…4 Scheme 1.3. The half reactions for denitrification. The first step is catalyzed by a variety of enzymes, depending on the organism, the second step is catalyzed by various types of nitrite reductase (NiR, Fig. 1.1), the third by nitric oxide reductases (NorB, Fig. 1.1), and the fourth by nitrous oxide reductase (NosZ, Fig. 1.1)……………………………………………………….5 Scheme 1.4. The half reactions for nitrate ammonification. The first step is catalyzed by a variety of enzymes, depending on the organism. In bacteria the second step is catalyzed by the enzyme cytochrome c nitrite reductase (ccNiR), which is the major topic of this thesis. Plants also have nitrite reductases capable of reducing nitrite directly to ammonia, but these enzymes are structurally quite different from the bacterial ones………………………………………...6 Scheme 1.5. The reactions for the anammox (anaerobic ammonia oxidation) process. 26, 27, 29 NO⋅ is generated from nitrite by specialized nitrite reductases………………………………………8 Scheme 1.6. Reactions catalyzed by ccNiR under standard assay conditions. The strong reductant methyl viologen monocation radical, MVred, is used as the electron donor, typically in concentrations of 80-100 µM………………………………………………………………….14 Scheme 1.7. Ambiguity associated with electronic structures in iron nitrosyl complexes, and the Enemark-Feltham notation used to deal with it (see text for details)……………………………20 Scheme 2.1. Nernst equation used to fit the Fig. 2.1 data. Here Cox refers to fully oxidized ccNiR, and Cred to the enzyme after a single reduction by n electrons. The remaining parameters are as defined in the main text…………………………………………………………………..35 Scheme 2.2. Chemical interpretation of the nitrite-loaded ccNiR 2-electron reduction. FeH1 represents the active site heme center in various states of oxidation and ligation; see text for details…………………………………………………………………………………………38 xvi Scheme 2.3. Here FeH1 represents the active site heme center in various states of oxidation and ligation. The solid arrows show the net reduction observed experimentally, while the dashed ones show the steps that likely underlie the observed process; see text for details………….51 Scheme 2.4. In the experiments described herein active site reduction in the presence of nitrite appears to proceed via Path 1. Under physiological conditions it will likely proceed via Path 2. In either case free NO⋅ release from subsitutionally labile {FeH1NO} 6 will be minimized if the standard reduction potential for reduction of {FeH1NO} 6 to {FeH1NO} 7 is much higher than that for reduction of FeH1 III (NO2 −) to {FeH1NO} 6 …………………………………………………52 Scheme 3.1. CcNiR-catalyzed reduction of nitrite to NO⋅ by ferrocyanide…………………..63 Scheme 4.1. Methyl viologen accepts an electron to produce MVred, a powerful reductant…..83 Scheme 4.2. Monomer-dimer equilibrium of methyl vilogen……………………………..…..85 Scheme A1. Equilibrium for nitrite binding with ccNiR…………………………………..…102 xvii