Single crystal spectroscopy and multiple structures from one crystal (MSOX) define catalysis in copper nitrite reductases

Abstract

Many enzymes utilize redox-coupled centers for performing catalysis where these centers are used to control and regulate the transfer of electrons required for catalysis, whose untimely delivery can lead to a state incapable of binding the substrate, i.e., a dead-end enzyme. Copper nitrite reductases (CuNiRs), which catalyze the reduction of nitrite to nitric oxide (NO), have proven to be a good model system for studying these complex processes including proton-coupled electron transfer (ET) and their orchestration for substrate binding/utilization. Recently, a two-domain CuNiR from a Rhizobia species (Br^2DNiR) has been discovered with a substantially lower enzymatic activity where the catalytic type-2 Cu (T2Cu) site is occupied by two water molecules requiring their displacement for the substrate nitrite to bind. Single crystal spectroscopy combined with MSOX (multiple structures from one crystal) for both the as-isolated and nitrite-soaked crystals clearly demonstrate that inter-Cu ET within the coupled T1Cu-T2Cu redox system is heavily gated. Laser-flash photolysis and optical spectroscopy showed rapid ET from photoexcited NADH to the T1Cu center but little or no inter-Cu ET in the absence of nitrite. Furthermore, incomplete reoxidation of the T1Cu site (∼20% electrons transferred) was observed in the presence of nitrite, consistent with a slow formation of NO species in the serial structures of the MSOX movie obtained from the nitrite-soaked crystal, which is likely to be responsible for the lower activity of this CuNiR. Our approach is of direct relevance for studying redox reactions in a wide range of biological systems including metalloproteins that make up at least 30% of all proteins.

Keywords: catalysis; electron transfer; metalloproteins; reaction intermediates; substrate utilization.

Fig. 1.

T2Cu site during the MSOX series of Br^2DNiR in an as-isolated state and optical spectra of the Br^2DNiR crystal for the duration of MSOX movie. (A) Optical spectra for a single crystal of blue Br^2DNiR at 100 K. A peak at 460 nm and large peak at 595 nm prior to X-ray exposure is characteristic of a blue CuNiR with oxidized T1Cu^II (solid blue line). Exposure to 0.4-MGy X-rays results (corresponding to DS1, C) in a complete loss of the peak at 460 nm and a substantial reduction of the peak at 595 nm (first dotted blue line). Further exposure to 0.4-MGy X-rays per dataset up to a total dose of 8 MGy (corresponding to DS20, E) results in the sequential loss of the 595-nm peak (dotted blue lines). Optical spectra for a single colorless dithionite-reduced crystal prior to X-ray exposure is shown and is characteristic of reduced T1Cu^I (solid red line). (B) Dose-dependent reduction of the peak at 595 in A and color change of crystal from intense blue (prior to X-ray exposure) to colorless (after a final total dose of 8 MGy). (C) The T2Cu site after first exposure to 0.4-MGy X-rays (DS1) showing two full occupancy waters (W1 and W2) coordinated to T2Cu, Asp92 in proximal position and two positions of a channel water (W4). Ile252 and His250 show no changes. (D) The T2Cu site in DS5, after exposure to 2-MGy X-rays, shows only partial density for W2 consistent with its gradual loss, while W1 still has full occupancy. No other changes are seen. (E) The T2Cu site in the final dataset (DS20), after exposure to 8-MGy X-rays, showing W2 completely disappeared and W1 still had full occupancy. No other changes are seen. 2F_o − F_c electron density maps for residues and waters are contoured at 1σ level. T2Cu is shown as a blue sphere. Occupancy of T2Cu is similar to T1Cu. Both are judged to be >0.9 by comparison of their B-factors to their coordinating protein ligands.

Fig. 2.

Time-resolved absorbance changes upon laser excitation for as-isolated Br^2DNiR and its comparison with as-isolated AxNiR. The initial rapid decrease in absorption at 595 nm is indicative of the reduction of the T1Cu by electrons generated by the photoexcitation of NADH. The slower restorative phase arises from the reoxidation of the T1Cu as ET to the T2Cu occurs. Note that no inter-Cu ET is observed in Br^2DNiR (black curve), while recovery of the 595-nm (T1Cu) optical signal is observed in AxNiR, consistent with ET to the T2Cu in AxNiR.

Fig. 3.

T2Cu site during the MSOX series of a nitrite-soaked Br^2DNiR crystal. (A) The T2Cu site after first exposure to 0.8-MGy X-rays (DS1) showing full occupancy of a single side-on nitrite coordinated to T2Cu, Asp92 in proximal position, and two channel waters (W4 and W5). Ile252 and His250 show no changes. (B) The T2Cu site in DS8 (6.4 MGy) showing equal occupancies of nitrite and NO. No other changes are seen. (C) The T2Cu site in DS17 (13.6 MGy) showing full occupancy of a single side-on NO coordinated to T2Cu. W4 has now disappeared. (D) The T2Cu site in DS25 (20 MGy) showing equal occupancies of NO and water (Wa). W4 has now returned. (E) The T2Cu site in DS38 (30.4 MGy) showing full occupancy of a single water coordinated to T2Cu, mimicking the oxidized T2CuII site in other prototypic CuNiRs. No other changes are seen. (F) The T2Cu site in the final dataset of the nitrite-bound MSOX series (DS65), after a total of 50 MGy, showing the single water (Wa) still coordinated to T2Cu. Asp92 shows signs of burning off due to dose limit in the crystal being exceeded with a loss of density observed. W4 and W5 are also almost completely disappeared. 2Fo − Fc electron density maps of residues are contoured at 1σ level. 2Fo − Fc electron density maps of ligands are contoured at 0.9σ level. T2Cu is shown as a blue sphere.

Fig. 4.

Time-resolved absorbance changes upon laser excitation in the presence of nitrite, absolute absorbance spectra before and after laser excitation, and online optical spectra of single nitrite-bound crystal before and after X-ray exposure used for a dataset for one structure. (A) Absolute absorbance spectra of Br^2DNiR in solution in the absence and presence of nitrite observed before and after laser excitation in solution. (B) Online optical spectra in a nitrite-soaked crystal of Br^2DNiR before and after X-ray exposure (0.8 MGy). (C) Absorbance changes at 595 nm in the presence of excess nitrite (10 mM) observed in the laser flash-photolysis instrument during the reduction of Br^2DNiR and its comparison with equivalent data for AxNiR. Only ∼20% recovery of the 595-nm band is observed in the case Br^2DNiR, indicating a lower degree of ET from T1Cu to T2Cu compared to AxNiR. For fuller experimental details of AxNiR, see ref. .