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. 2019 Jul 5;9(7):6087-6099.
doi: 10.1021/acscatal.9b01266. Epub 2019 May 29.

Unexpected Roles of a Tether Harboring a Tyrosine Gatekeeper Residue in Modular Nitrite Reductase Catalysis

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Free PMC article

Unexpected Roles of a Tether Harboring a Tyrosine Gatekeeper Residue in Modular Nitrite Reductase Catalysis

Tobias M Hedison et al. ACS Catal. .
Free PMC article

Abstract

It is generally assumed that tethering enhances rates of electron harvesting and delivery to active sites in multidomain enzymes by proximity and sampling mechanisms. Here, we explore this idea in a tethered 3-domain, trimeric copper-containing nitrite reductase. By reverse engineering, we find that tethering does not enhance the rate of electron delivery from its pendant cytochrome c to the catalytic copper-containing core. Using a linker that harbors a gatekeeper tyrosine in a nitrite access channel, the tethered haem domain enables catalysis by other mechanisms. Tethering communicates the redox state of the haem to the distant T2Cu center that helps initiate substrate binding for catalysis. It also tunes copper reduction potentials, suppresses reductive enzyme inactivation, enhances enzyme affinity for substrate, and promotes intercopper electron transfer. Tethering has multiple unanticipated beneficial roles, the combination of which fine-tunes function beyond simplistic mechanisms expected from proximity and restrictive sampling models.

Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Rationale and strategy for studying the role of redox partner tethering in copper containing nitrite reductase. (A) Structure (in complex with Ax cytochrome c551; PDB ID 2ZON; top) and proposed mechanism (bottom) of prototypic 2-domain AxNiR. (B) Structure (PDB ID: 3ZIY; top) and proposed mechanism (bottom) of the 3-domain copper containing nitrite reductase, RpNiR, used in this study. In (A) and (B), the three monomers in the structure of the trimeric CuNiRs are shown as green, magenta, and cyan. The isolated cyt c551 protein is shown in yellow. (C) Strategy of dissecting the 3-domain cytochrome c-tethered Ralstonia pickettii copper nitrite reductase into the component domains. In the schematic shown in (C), the three monomers in the structure of RpNiR are shown as green, magenta, and cyan. The isolated RpNiR cyt c protein is shown in yellow.
Figure 2
Figure 2
Structural organization of the RpNiR-core trimer. (A) Cartoon representation of the trimeric RpNiR-core protein (viewed along the 3-fold axis). (B) A close-up view (bottom) of the RpNiR-core protein showing details of the Tyr323 site. In (A) and (B), the monomeric units of the RpNiR-core protein are colored in green, magenta, and cyan. Copper ions are shown as dark blue spheres. (C) Comparison between the RpNiR-core protein (lilac) and the full-length native RpNiR (salmon), showing details of the differences in the linker region (residues 315–333). (D) A close-up view of the T2Cu site and residue Ile245, found in the channel from the T2Cu site toward the surface of the protein in the RpNiR-core protein (lilac and gray). (E) Alignment of the RpNiR-core trimer (lilac and gray) with the full-length RpNiR (PDB ID: 3ZIY; salmon). (F) Alignment of the RpNiR-core trimer (lilac and gray) with PhNiR (PDB ID: 2ZOO; yellow).
Figure 3
Figure 3
Details of the T2Cu sites of the RpNiR-core proteins and the full-length RpNiR variant proteins in “as-isolated” and “nitrite-bound” form. (A) Water ligands, W1 and W2, bound to T2Cu in the “as-isolated” RpNiR-core protein. (B) Nitrite bound to T2Cu in the top-hat” and (C) “side-on” conformations in the RpNiR-core protein (see Figure S4 for the stereo view). At the T2Cu site, W1 is coordinated to the T2Cu and W2 is hydrogen bonded to W1. (D), (E), and (F) show structures of “as-isolated” full-length RpNiR (PDB ID: 3ZIY), Y323A, and Y323E variants, respectively. (G), (H), and (I) show the “nitrite-bound” forms of Y323F, Y323A, and Y323E variants, respectively (see Figure S4 for the stereo view). Hydrogen bonds are shown as black dotted lines, coordination bonds are in red, copper ions are shown as blue spheres, and water molecules are small red spheres. A 2Fo-Fc electron density map is contoured at the 1.0σ level for the T1Cu and T2Cu sites and shown as light-gray mesh.
Figure 4
Figure 4
Domain conformational dynamics limit haem to T1Cu electron transfer in RpNiR. (A) Cartoon representation of the RpNiR structure showing a close-up of the haem and T1Cu centers. (B) Example laser flash photolysis transient for intraprotein electron transfer reaction from the haem to the T1Cu in nitrite-free RpNiR. The red transient shows the background reaction associated with NADH photoexcitation in the presence of the mediator, N-methyl nicotinamide (NMN); the black transient shows the reaction with the RpNiR protein present in the reaction mixture; and the blue transient is the deconvoluted trace, corresponding to the haem to T1Cu electron transfer step in RpNiR. (C) Comparison of experimental small-angle X-ray scattering (SAXS) from full-length wild-type RpNiR with that calculated from the crystal structure (PDB ID: 4AX3), depicted as inset, χ2 = 21.0. (D) Comparison of experimental SAXS data with that calculated for a model, shown as inset, of RpNiR created using the linker conformation of the RpNiR-core structure and refining the position of conformationally plastic cyt c domains, χ2 = 1.5. (E) A comparison of SAXS distance distribution functions (P(R)) for compact and elongated RpNiR. (F) The rate of interprotein electron transfer from the isolated cytochrome c protein to the core CuNiR proteins.
Figure 5
Figure 5
Nitrite binding and redox communication between the tethered haem domain and the catalytic T2Cu in RpNiR. (A) EPR spectra of the different RpNiR constructs with and without 5 mM nitrite present. In (A), the WT RpNiR spectra is shown in black and the RpNiR-core is shown in red. (B) Steady state Michaelis–Menten plots of the different CuNiR constructs (WT AxNiR is shown as red circles; WT RpNiR is shown as black circles; RpNiR-core is shown as black squares; and Y323F RpNiR-core is shown as black diamonds). The kinetic parameters obtained are shown in Table 4. (C) UV–vis spectra of oxidized (black) and 1-electron reduced (red) WT RpNiR, showing how 1-electron predominately sits on the haem cofactor. (D) Effect of titrating nitrite into 1-electron reduced WT RpNiR measured by the absorbance of the haem Soret peak. The insert in (D) shows the observed rate constants for the reaction between 1-electron reduced RpNiR and nitrite. The Ks value for nitrite binding measured by UV–vis spectral titration is 3.7 × 10–3 ± 0.3 × 10–3 mM, and the Ks and klim values for the nitrite dependent stopped-flow are 3.5 × 10–3 ± 1.2 × 10–3 mM and 0.34 ± 0.02 s–1, respectively.

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