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.

Figure 1

Architecture of the ccNiR active site. (A) Active site of S. oneidensis cytochrome c nitrite reductase. (B) Active site of nitrite-bound Wolinella succinogenes ccNiR showing the hydrogen bonding network. Key active site residues are colored green. Residues are numbered using S. oneidensis ccNiR numbering. Images generated in PyMol from Protein Data Bank entries 3UBR and 2E80, respectively.

Scheme 1. Proposed Reaction Scheme for the Six-Electron Seven-Proton Reduction of Nitrite to Ammonia by ccNiR

Proposed radical intermediates are denoted with asterisks. Residues predicted to be involved in steps are denoted. Adapted from ref (25).

Figure 2

Current–potential profiles of (A) WT ccNiR and its (B) Y206F, (C) R103K, and (D) R103Q mutants. All experiments were performed at pH 7, 20 mV/s, and 20 °C, with an electrode rotation rate of 3000 rpm. Top panels show overlays of raw cyclic voltammograms recorded at increasing nitrite concentrations. Dotted cyclic voltammograms show data of ccNiR films recorded in the absence of nitrite. Bottom panels show first derivatives of baseline-subtracted reductive scans of the voltammograms. Insets show plots of E_cat values vs nitrite concentration fit to eq 3. Substrate concentrations were 16–473 μM for WT, 2–120 μM for Y206F, 16 μM to 1.9 mM for R103K, and 76 μM to 8.4 mM for R103Q.

Figure 3

Dependence of the ccNiR catalytic waveform on pH. All scans were recorded at 500 μM nitrite, 20 mV/s, and 20 °C, with an electrode rotation rate of 3000 rpm. Top panels show raw voltammograms. Bottom panels show first derivatives of baseline-subtracted reductive scans. The cell solution pH is shown at the top left of each panel.

Figure 4

pH dependence of catalytic features during WT and mutant ccNiR nitrite turnover. (A) Plot of E_cat1 (○) and E_cat2 (◆) for WT ccNiR. (B) Plots of E_cat2 values for WT (◆), Y206F (□), R103Q (■), and R103K (▲). Experiments were conducted at 500 μM nitrite, 20 °C, and 20 mV/s, with an electrode rotation rate of 3000 rpm. E_cat2 data for WT, R103Q, and R103K are fit to eq 1, and E_cat2 data for Y206F are fit to eq 2.