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 has been assumed that this is also true in vivo. However, this report demonstrates that it is possible to trap a putative intermediate by controlling the electrochemical potential at which reduction takes place. UV/vis spectropotentiometry showed that nitrite-loaded Shewanella oneidensis ccNiR is reduced in a concerted two-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 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 does not accumulate, while at higher potentials it is trapped, thus preventing catalytic ammonia formation.

The decaheme enzyme cytochrome c nitrite reductase (ccNiR) catalyzes reduction of nitrite to ammonium in a six-electron, eight-proton process. With a strong reductant as the electron source, ammonium is the sole product. However, intermediates accumulate when weaker reductants are employed, facilitating study of the ccNiR mechanism. Herein, the early stages of Shewanella oneidensis ccNiR-catalyzed nitrite reduction were investigated by using the weak reductants N,N,N′,N'-tetramethyl-pphenylenediamine (TMPD) and ferrocyanide. In stopped-flow experiments, reduction of nitrite-loaded ccNiR by TMPD generated

Cytochrome c Nitrite Reductase (ccNiR) is a periplasmic homodimeric decaheme enzyme that catalyzes the reduction of nitrite to ammonium in a process that involves six electrons and eight protons. Under standard assay conditions, which use a strong reducing agent as an electron source, catalysis takes place rapidly without producing detectable intermediates. However, intermediates do accumulate when weaker reducing agents are employed, allowing the ccNiR mechanism to be studied. Herein, the early stages of Shewanella oneidensis ccNiR‐catalyzed nitrite reduction were investigated in isolation by using the weak reducing agents N,N,N’,N’‐tetramethyl‐p‐phenylenediamine (TMPD) and the 2‐electron reduced form of indigo trisulfonate. Experiments were done with the wild type enzyme (wtccNiR) as well as with R103Q, Y206F, and H257Q variants. A UV/Vis stopped‐flow investigation of the reaction between nitrite‐loaded wtccNiR and TMPD revealed for the first time that the reaction proceeds via a transient 1‐electron reduced intermediate. Generation of this species is pH‐independent, whereas its decay to a previously characterized 2‐electron reduced intermediate is fastest at pH 6.8 and significantly slower at higher pH. The pH dependence is ascribed to the rate‐limiting cleavage of the nitrite N – O bond, which requires a prior di‐protonation predicted to be slow in earlier computational studies. Under steady‐state conditions, S. oneidensis ccNiR catalyzed the slow 1‐electron reduction of nitrite to nitric oxide, which was monitored by tracking the concomitant appearance of the colored TMPD+ radical product. The rate of TMPD+ formation was found to be directly proportional to the concentration of TMPD+ at low TMPD concentrations, providing important insights about the mechanism of NO˙ release. The steady‐state studies also showed that nitrite is a substrate inhibitor of NO˙ release when TMPD is the electron source, probably because it blocks exit by NO˙ through the nitrite entry channel. The H257Q variant of ccNiR was found to have 1/400th of the wild type enzyme's nitrite reductase activity in the standard assay in which methyl viologen monocation radical is the electron source, but nearly normal hydroxylamine reductase activity. This demonstrated that H257 is essential for nitrite reduction but not for reduction of hydroxylamine, a putative intermediate in the catalytic process. UV/Vis spectropotentiometry showed that the nitrite‐loaded active site of H257Q still reduced at fairly high applied potential, but the reduction was by one electron, whereas the wild type is reduced in a concerted 2‐electron step. The experiments with the variant, coupled with the pH‐dependent stopped flow experiments with wtccNiR, confirm H257's importance in facilitating nitrite reduction, but suggest that its role is to modulate the pKa values of one or more of the waters that form a complex hydrogen bonding network in the active site. This view is more conservative than earlier theoretical predictions of direct histidine involvement in nitrite d...

Details of current and previous biologic therapy and concurrent immunosuppression was recorded. Results 158 patients (69.6% Crohn's disease (CD) 40.4% ulcerative colitis (UC)) required biologic treatment (BT) for IBD. 51.8% of these patients stopped BT over this time period. The median duration of BT was 16 months . Of these 60 and 11 patients required a second and third agent respectively. 96.2% of patients who stopped BT were on an anti-TNF agent (p=0.001). The reasons for changing therapies are detailed in Figure 1. Patients with CD were 2.82 times (95%CI 1.09-7.33, p=0.033) more likely to lose response to biological therapy than patients with UC. The odds ratio of losing response to BT in those patients on infliximab compared with alternative agents was 4.11 (95%CI 0.89-19.01, p=0.071). Patients on infliximab were 6.39 times (95%CI 0.80-51.13, p=0.081) more likely to lose response to BT than those on adalimumab. There were no other significant differences between biological agents and other reasons for stopping therapy.32 cases (55.1%) were identified as having discontinued treatment due to clinical remission. Of these, 12 cases were thiopurine naïve previously. 24 cases had been followed up for over 12 months since discontinuation. The within 12 month relapse rate was 37.5% and the median time to relapse was 9 months (IQR 6-13.5). 93.3% of patients who relapsed were established on an immunomodulator prior to remission. There was no significant difference in the rate of relapse between step-up vs step-down therapy (p=0.647). Fifteen cases were re-challenged with the same biologic for relapse after successful remission, 80% of whom had a successful rechallenge. Conclusions Findings in our centre are comparable to the recognised rate of patients relapsing within a year of achieving remission and discontinuing therapy. There was no significant difference in the rate of relapse in patients who achieved remission between those who were stepped up or stepped down to biologics. The higher proportion of patients who lost response to infliximab may be a consequence of the drug's higher immunogenicity.

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 has been assumed that this is also true in vivo. However, in vitro we have found it possible to trap putative intermediates, or to release partially reduced nitrogen species such as nitric oxide, by controlling the electrochemical potential at which reduction takes place. This poster will present UV‐Vis spectropotentiometric titrations of the active site variants R103Q and H257Q, and steady‐state and stopped‐flow kinetic studies of ccNiR‐catalyzed reduction of nitrite to nitric oxide by the weak reductant N,N,N′,N′‐tetramethyl‐p‐phenylenediamine (TMPD). Wild type nitrite‐loaded ccNiR is reduced chemically or electrochemically at very high potentials (midpoint potential of 246 mV vs SHE) in a concerted 2‐electron process that forms an {FeNO}7 moiety at the active site. By contrast, the H257Q variant’s active site midpoint potential is downshifted to 125 mV vs SHE and is now a 1‐electron reduction to yield an {FeNO}6 moiety. The wild type catalyzes the reduction of nitrite to NO• by TMPD. H257Q does not appear to catalyze this reaction, which may be due to its lower active site midpoint potential or to the much slower turnover of the mutant; studies with more powerful reductants are now under way.