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

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.

Redox reactions are an essential component in a wide range of biological systems, most notably in respiration (1) and photosynthesis (2). These critical reactions are often performed by metal-containing systems including metalloproteins that form a large portion of the protein kingdom. It is estimated that one-third of all proteins in nature require metals to perform their biological roles and nearly one-half of all enzymes must associate with a particular metal to function (3, 4). These metal ions can be either a single atom or combined with other atoms to form part of a cluster, playing a variety of life sustaining roles in the bacterial, plant, and animal kingdoms. They exploit the oxidation states of metals to perform redox cycling during catalysis and include metalloenzymes such as cytochrome c oxidase, hydrogenases, nitrogenases, and nitrite reductases where catalysis involves the controlled delivery of electrons and protons to the active site for the utilization of chemical substrates. Most of these systems utilize multiple redox centers among which these catalytic events are often coordinated, coupled, and orchestrated by structural signals that remain poorly understood. The redox-active metalloenzyme copper nitrite reductase (CuNiR) has become a good model system for studying these complex processes in biological systems with coupled redox centers due to their amenability to spectroscopic, fast kinetic, and advanced structural approaches capable of providing a movie of catalytic reaction activated by electron transfer (ET) in crystallo between the coupled centers and its utilization for the conversion of substrate providing information on reaction intermediates, some of which may be transitory (5–9).CuNiRs catalyze the conversion of nitrite to gaseous nitric oxide (NO₂⁻ + 2H⁺ + e⁻ → NO + H₂O) in the first committed step of the denitrification pathway. This highly conserved family of enzymes is widespread in nature (10) and is of major importance in several pathways in the biogeochemical nitrogen cycle (11, 12). These homo-trimeric proteins contain two types of redox Cu center per monomer, as follows: an electron accepting type-1 Cu (T1Cu) site, which receives an electron from a physiological redox partner (cytochrome c or pseudoazurin/azurin), and a catalytic type-2 Cu (T2Cu) site. The T1Cu site is located near the top of the monomer and is responsible for giving rise to the color of the enzyme (blue or green) in its oxidized Cu^II] state, depending on subtle differences in the immediate coordination chemistry of the site. The T2Cu is located within the interface of two adjacent monomers and is responsible for the binding of nitrite and its catalysis. The two redox Cu centers are coupled via the neighboring Cys-His residues that form a conserved hard-wired 12.6-Å ET bridge. When T2Cu is in the oxidized substrate-free Cu^II] state, with a Cu^II-(His)₃-H₂O coordination, the difference between the reduction potentials of the two Cu sites are small or energetically unfavorable to allow ET from T1Cu to T2Cu. Displacement of the coordinated water by nitrite increases the reduction potential of T2Cu and promotes inter-Cu ET, an event heavily gated by the provision of protons from two conserved residues (His_CAT and Asp_CAT) in the T2Cu pocket to the substrate (5, 13). In the CuNiR of Achromobacter xylosoxidans (AxNiR), laser flash photolysis had shown the rate of inter-Cu ET to be the same as the rate of proton uptake, providing clear evidence for proton-coupled ET (PCET). Following the reduction of nitrite to nitric oxide (NO), the product dissociates from the T2Cu site and water rebinds to return to the resting T2Cu^II state. A delicate and ordered mechanism ensures a “dead-end” inactive species is not formed where the T2Cu redox center is in a reduced Cu^I] state with the water ligand disociated, that is then unable to bind the substrate (14, 15). This mechanism for CuNiR is supported by spectroscopic, kinetic, mutagenesis, and structural studies primarily from the well-studied blue AxNiR and the green CuNiRs of Achromobacter cycloclastes (AcNiR) and Alcaligenes faecalis (AfNiR) (13–18).Biological systems containing redox centers, including metalloproteins such as CuNiR, are prone to reduction from X-ray sources during data collection (19, 20) due to solvated electrons within the crystal being produced from radiolysis of waters that can rapidly reduce their primary redox centers, e.g., T1Cu in CuNiRs is reduced from Cu^II to Cu^I redox state but the T2Cu remains oxidized (17). This phenomenon has been exploited to initiate enzyme turnover to generate reaction intermediates through redox driven catalysis (21). We have developed the MSOX (multiple structures from one crystal) approach that enables the collection of several structures from the same spot in a crystal (6–8). This approach has been applied successfully to AcNiR at several temperatures to provide structural movies of the catalytic reaction in nitrite-soaked crystals. However, these studies have not provided any information on the redox state of metal centers during catalysis in the crystals.A two-domain blue CuNiR from a Rhizobia species (Br^2DNiR) has recently been structurally characterized showing an unusual oxidized T2Cu^II-(His)₃-(H₂O)₂ coordination site, requiring the displacement of two water molecules by the substrate (22, 23) instead of a single water molecule in a prototypic CuNiR (15, 24). The recent availability of highly diffracting crystals of Br^2DNiR and the ability to achieve full occupancy of nitrite at the T2Cu site in nitrite-soaked crystals (22) prompted us to utilize the MSOX approach to probe structural changes during enzyme turnover. MSOX of as-isolated crystals of Br^2DNiR was combined with the recently commissioned on-line single crystal optical spectroscopy at SPring-8 BL26B1, enabling the redox status of the optically visible T1Cu to be monitored during serially recorded multiple structures and reporting on the structural changes at the catalytic T2Cu site for a CuNiR in the resting state. This has enabled us to provide a spectroscopically validated MSOX movie of an as-isolated CuNiR. We have combined detailed structural observations recorded in these MSOX movies and single crystal spectroscopy with solution measurements of reduction potentials and inter-Cu ET using laser-flash photolysis. We provide unambiguous evidence for a strong gating of ET between the coupled redox centers that is removed in the presence of the substrate. We demonstrate that the experimental approach (marrying single crystal spectroscopy/MSOX with solution data) utilized here is powerful in dissecting complex redox reactions and suggest the approach should be applicable to many complex redox systems in biology.