Hydrogen Bonding Networks Tune Proton-Coupled Redox Steps during the Enzymatic Six-Electron Conversion of Nitrite to Ammonia

Abstract

Multielectron multiproton reactions play an important role in both biological systems and chemical reactions involved in energy storage and manipulation. A key strategy employed by nature in achieving such complex chemistry is the use of proton-coupled redox steps. Cytochrome c nitrite reductase (ccNiR) catalyzes the six-electron seven-proton reduction of nitrite to ammonia. While a catalytic mechanism for ccNiR has been proposed on the basis of studies combining computation and crystallography, there have been few studies directly addressing the nature of the proton-coupled events that are predicted to occur along the nitrite reduction pathway. Here we use protein film voltammetry to directly interrogate the proton-coupled steps that occur during nitrite reduction by ccNiR. We find that conversion of nitrite to ammonia by ccNiR adsorbed to graphite electrodes is defined by two distinct phases; one is proton-coupled, and the other is not. Mutation of key active site residues (H257, R103, and Y206) modulates these phases and specifically alters the properties of the detected proton-dependent step but does not inhibit the ability of ccNiR to conduct the full reduction of nitrite to ammonia. We conclude that the active site residues examined are responsible for tuning the protonation steps that occur during catalysis, likely through an extensive hydrogen bonding network, but are not necessarily required for the reaction to proceed. These results provide important insight into how enzymes can specifically tune proton- and electron transfer steps to achieve high turnover numbers in a physiological pH range.

De novo design of protein homo-oligomers with modular hydrogen-bond network–mediated specificity

“…The DNA double helix elegantly resolves both challenges; paired bases come together such that all buried polar atoms make hydrogen bonds that are self-contained between the two bases and have near ideal geometry. In proteins, meeting these challenges is more complicated because backbone geometry is highly variable and pairs of polar amino acids cannot generally interact as to fully satisfy their mutual hydrogen bonding capabilities; hence sidechain hydrogen bonding usually involves networks of multiple amino acids with variable geometry and composition, and there are generally very different networks at different sites within a single protein or interface pre-organizing polar residues for binding and catalysis {Chakrabarti:2005jw, Judd:2014io, SanchezAzqueta:2014gt} {Xu:1997wr, Sheinerman:2002hi}.…”

Correlations between the Electronic Properties of Shewanella oneidensis Cytochrome c Nitrite Reductase (ccNiR) and Its Structure: Effects of Heme Oxidation State and Active Site Ligation

“…The mutation was verified by sequencing. Transformed cells were grown in 1L batches of LB medium at 30°C for 24 hours, after which ccNiR H268M was purified using a Ni-6-Sepharose column (GE Healthcare), and the His tag removed by overnight digestion with recombinant TEV protease, as described previously (22) .…”

Modelling the voltammetry of adsorbed enzymes and molecular catalysts

“…The steady-state response sometimes exhibits a sigmoidal current boost or decrease that is superimposed onto the "main" catalytic response [37], as recently illustrated in studies of flavo- [38], molybdo- [39] or heme-enzymes [40][41][42]. This may result from allosteric effects: active site chemistry and/or intramolecular ET kinetics may be affected by the redox state of a center that is remote from the active site.…”