Active Intermediates in Copper Nitrite Reductase Reactions Probed by a Cryotrapping‐Electron Paramagnetic Resonance Approach

Abstract Redox active metalloenzymes catalyse a range of biochemical processes essential for life. However, due to their complex reaction mechanisms, and often, their poor optical signals, detailed mechanistic understandings of them are limited. Here, we develop a cryoreduction approach coupled to electron paramagnetic resonance measurements to study electron transfer between the copper centers in the copper nitrite reductase (CuNiR) family of enzymes. Unlike alternative methods used to study electron transfer reactions, the cryoreduction approach presented here allows observation of the redox state of both metal centers, a direct read‐out of electron transfer, determines the presence of the substrate/product in the active site and shows the importance of protein motion in inter‐copper electron transfer catalyzed by CuNiRs. Cryoreduction‐EPR is broadly applicable for the study of electron transfer in other redox enzymes and paves the way to explore transient states in multiple redox‐center containing proteins (homo and hetero metal ions).


Introduction
Tr ansition metals (e.g.Cu, Mn, Fe and Mo) are ubiquitous in biology and play key roles in redox enzymes that are vital for life and the next generation of biofuels. [1] Due to their complex reaction mechanism, and often, poor optical signals (e.g. the weak absorbance bands from the type-II copper centers [2] ), identifying and studying active intermediates in the reaction cycle of metal containing enzymes is challenging. Methods must be established for deeper mechanistic insights into these enzymes.H ere,w ed evelop the use of cryoreduction (with annealing at higher temperatures) in combination with electron paramagnetic resonance (EPR) spectroscopy for monitoring and detecting active intermediates in the electron transfer reactions of complex metalloenzymes.
Cryoreduction-EPR involves the use of ionizing radiation to reduce frozen solutions of protein (e.g.7 7K). Then, by annealing the frozen protein solution to higher temperatures, reaction intermediates are formed and trapped, enabling the capture of transient enzyme species,w hich if paramagnetic, can be investigated using EPR spectroscopy.Active catalytic intermediates in an umber of enzymes containing heme catalytic centers [3][4][5] have been identified using similar cryoreduction-EPR methods (e.g.i ni dentification of ferrichydroperoxo,f erric-peroxo,f errous-superoxo radical and compound Ii nt he P450 monooxygenase reaction cycle). [3] DNAr adicals have also been investigated using cryoreduction-EPR methods. [6,7] In this study,a sap roof-of-principle, cryoreduction-EPR is used to detect substrate binding, product release and the active intermediates when electrons are transferred from the type-I to the type-II copper centers in the copper-containing nitrite reductase (CuNiR) family of enzymes.
Here,w eu sed a 60 Co source of U-radiation at 77 Kt o cryolytically reduce the copper centers in two coppercontaining nitrite reductases,t he two-domain AxNiR and the core portion of the three-domain RpNiR, in the presence and absence of the substrate,n itrite ( Figure 1). Using annealing and EPR spectroscopy,s imultaneous tracking of T1 and T2Cu redox centers was used to follow and probe inter-copper electron transfer.Initially,weperformed studies on "nitrite-free" forms of the well-characterized two-domain AxNiR and the RpNiR core protein for which an X-ray crystal structure and solution properties have been reported. [15] Results and Discussion In Figure 2A,t he EPR spectra of the oxidized "nitritefree" AxNiR and RpNiR core proteins measured at 20 Kare shown. These spectra display the presence of the overlapping, four-line,p arallel hyperfine features,a rising from both the T1Cu and T2Cu centers.F rom experimental and simulated EPR spectra (see Figure S1) collected on the RpNiR core protein, it is observed that the EPR signals at the perpendicular orientation is split into four hyperfine lines,afeature that is due to the strong hyperfine coupling of the 63, 65 Cu nuclei with the electron spin of T1Cu center.T his splitting is absent in the "nitrite free" AxNiR sample,s uggesting subtle differences in the electronic structures of the T1Cu centers present in both of these CuNiR proteins ( Figure S1 in the Supporting Information). [25] After irradiation of frozen AxNiR and RpNiR proteins at 77 Kw ith U-rays from a 60 Co source, areduction in the T1Cu EPR signal is observed (approx. 30 % and 50 %f or AxNiR and the RpNiR core protein, respec- Figure 1. Schematic of cryoreduction-EPR used to study inter-copper electron transfer in CuNiRs. g-irradiation at alow temperature (77 K) from a 60 Co source is used to selectively reduce the T1Cu site in CuNiRs (e.g. AxNiR;P DB ID:1OE1), and by incrementally raising and holding the temperature, electrons transfer from the T1 to the T2Cu centers. All EPR spectra were recorded at 20 K. Figure 2. T1 to T2Cu electron transfer monitored in "nitrite-free" AxNiR and RpNiR core proteins through the cryoreduction-EPR method. A) EPR spectra of oxidized( black, solid lines) and cryolyticallyr educed (red, dotted lines) AxNiR (bottom) and RpNiR core (top) proteins. ObservedE PR spectra when B) AxNiR and C) RpNiR core samples were annealed (left) and changes in EPR intensities of the T1 and T2Cu sites relative to the starting signal at 158 Kf or AxNiR and 170 K for RpNiR core proteins, respectively (right). All EPR spectra were recorded at 20 K. During the irradiation process, many paramagnetic EPR signals are produced and the signal indicated by the black asterisk mark in the RpNiR core sample is due to the [H] radical and is formed in all the samples examinedh ere, including both the buffer control and empty EPR quartz tubes ( Figure S6). [28,29] tively; Figure 2and  , aresult that is in-line with that observed in an in crystallo AxNiR study. [26] As previously suggested, the selective reduction of the T1 over the T2Cu center is likely due to the solvent accessibility of the T1Cu, which is situated 7 from the surface of the protein. [17] TheT2Cu of CuNiRs is buried within the trimeric core of the protein.
In Figure 2B,t emperature-dependent changes in the oxidized T1 and T2Cu EPR signals (measured at 20 K) for substrate-free AxNiR are presented. When AxNiR samples were annealed, the intensity of the signals attributed to the Cu II state of the T1Cu and the T2Cu lessened ( Figure 2B and Figure S2). Moreover,attemperatures lower than 200 K, only as mall fraction of the electrons are transferred from the T1Cu to the T2Cu (< 5% of the total T2Cu signal). The majority of T2Cu reduction occurs only above 200 K, atemperature that is generally regarded as the protein "glass transition" temperature. [27] We demonstrated recently that "solvent-slaved" protein motions assist AxNiR-catalyzed PCET reactions. [20] As the majority of protein motions are "frozen" below the "glass transition" temperature, [27] the EPR data presented here provide further support for arole of protein dynamics in AxNiR inter-copper electron transfer.
Solvated electrons generated from the U-radiation in principle could directly reduce the T1 and the T2Cu centers of AxNiR during the annealing process.Should this be the case, the annealing process would not report directly on T1 to T2Cu electron transfer. We therefore performed cryoreduction-EPR measurements on af orm of CuNiR that cannot transfer electrons between the copper centers in the absence of nitrite.A lthough the RpNiR core protein is structurally similar to the AxNiR protein ( Figure S4), it has recently been reported that it is unable to transfer electrons between the copper centers in the absence of bound nitrite. [15] This is attributed to the high T1Cu mid-point potential relative to the T2Cu in the substrate free form. [15] As such, the T2Cu of the RpNiR core protein should in theory remain oxidized during the annealing process.
Te mperature-dependent changes in the T1 and T2Cu signals of the cryolytically reduced RpNiR core samples are shown in Figure 2C.Unlike the AxNiR protein, there are few or no alterations in the intensity of the T2Cu signal of RpNiR core during temperature ramping (between 170 and 250 K; Figure S3), indicating that solvated electrons produced from U-radiation do not directly reduce the T2Cu of the CuNiR proteins during the annealing.W ed oh owever observe ac hange in the electronic properties of the T2Cu center during annealing, with as econd paramagnetic T2Cu species (T2Cu [2]) forming over the experimental temperature range ( Figure S5). Between 170-250 K, the signal attributed to the T1Cu continuously decreases until it plateaus at aminimum. Unexpectedly,b etween 250 and 270 K, we observe what we hypothesize to be electron transfer from the fully reduced T1Cu to the T2Cu [2] species,c ausing as hift in the T2Cu equilibrium position towards the "resting" T2Cu species (T2Cu [1]) accompanied by adecrease in the T2Cu hyperfine intensity ( Figure S5).
In our earlier studies,w er eported that T1 to T2Cu electron transfer was inhibited in the RpNiR core protein in absence of nitrite. [15] This hypothesis was based on recorded mid-point potentials and alaser flash photolysis assay,which was used to monitor changes in the UV/Vis active T1Cu site when laser pulses were used to rapidly inject the protein with electrons.U sing the EPR approach presented here to probe the T2Cu site,w eh ave been able to observe an additional previously uncharacterized T2Cu species,T2Cu [2],present in the RpNiR core protein, which is formed upon reduction of the T1Cu and can facilitate inter-copper electron transfer.W e must note that in our cryoreduction-EPR experiments performed on the RpNiR core protein, the percentage signal change attributed to the reduction of the T1Cu site during annealing is far lower than that seen in the AxNiR sample (approx. 15 %inRpNiR core and 40 %inthe AxNiR sample). We attribute this to al arger percentage of T1Cu reduced in the RpNiR core protein during the initial cryolytic reduction process (approx. 30 %a nd 50 %f or AxNiR and the RpNiR core protein, respectively; Figure 2and Figure S2,S3), aresult that is due to different amounts of exposed 60 Co-U irradiation on the samples (22 kGy in AxNiR and 50 kGy in RpNiR core), and also plausibly,aresult of redox potential differences for the T1Cu centers in the different constructs (+ 255 mV in AxNiR, [30] and + 331 mV in the RpNiR core [15] ).
We also performed cryolytic reduction-EPR measurements on "nitrite-bound" forms of AxNiR and the RpNiR core proteins.C ontinuous wave EPR spectra of "nitritebound" AxNiR and the RpNiR core proteins are shown in Figures 3A and C, respectively.A se xpected, in the AxNiR protein sample containing nitrite,the T2Cu, and not the T1Cu center is altered (A k ( 63,65 Cu;T 2); % 370 MHz with g = 2.290; AxNiR). This is indicative of the nitrite being bound to the catalytic T2Cu site.F or the "nitrite-bound" RpNiR core protein, there are subtle changes in the T2Cu hyperfine features.B ased on our new EPR spectral simulations (Figure S7), approx. 20 %o ft he T2Cu hyperfine features have shifted from a" nitrite-free" to a" nitrite-bound" state when the oxidized RpNiR core was incubated with 5mm nitrite.No additional changes in the hyperfine features of the T2Cu center were observed upon the addition of supplementary nitrite,s uggesting that in an oxidized state only af raction of the RpNiR core population can accept nitrite.P revious studies have shown that aconserved tyrosine residue,present on ac yt c linking region, blocks nitrite from binding to the T2Cu site of the oxidized full-length RpNiR protein. [15] This tyrosine residue is present in the RpNiR core protein, but occupies an alternative state in the X-ray structure,a llowing nitrite to bind. [15] We hypothesize that in solution, this linker and tyrosine residue may occupy multiple conformations, both blocking and allowing access of the nitrite substrate to the T2Cu center.
Te mperature dependent changes in the oxidized T1 and T2Cu EPR signals (Cu II )o f" nitrite-bound", cryolytically reduced AxNiR are presented in Figure 3B,F igure S2 (absolute EPR signal) and S8 (relative EPR signal). Like the "nitrite-free" samples,a nd indicative of the involvement of protein motions in inter-copper electron transfer of CuNiRs, there is little or no electron transfer below the "glass-transition" temperature ( Figure 3B,S 2a nd S8). However, when samples are annealed to 198 Ka nd then further to 218 K, there is an increase in the number of electrons transferred from the T1Cu to the T2Cu centers (shown by an approx. 30 %reduction in the T2Cu signal and an increase in the intensity of the T1Cu signal, Figure S2 and S8). There is no change in EPR line width, and only as light change in spectral line position of T2Cu EPR signal during this electron transfer process ( Figure 3B). Based on the spectroscopic properties of the species seen at 198 and 218 K, it is likely that it is associated with the reduction of nitrite to nitric oxide and the dissociation of the product from the active site.Ithas been proposed that nitrite reduction at T2Cu and release of the product, nitric oxide,i nvolves the consumption of two protons. [17,30] This would lead to the formation of either [Cu I ÀNO + ]or[Cu II ÀNO] redox state at the T2Cu, neither of which are EPR active.T he observation of ad ecrease in intensity in T2Cu EPR signal with A k ( 63, 65 Cu;T 2); % 370 MHz with g = 2.290 also rules out the formation of [Cu I ÀNO] redox state,w hich would be observed g 2.00 region, as in the case of Rhodobacter sphaeroides NiR (RsNiR), [31] and end-on bound Cu I ÀNO inorganic model complex, [32] with strong hyperfine coupling to the 14 [17,18,20] the proton coupled electron transfer to the nitrite-bound T2Cu center leads to nitrite reduction and release of the product, nitric oxide,w ith the formation of [Cu II À OH] redox state at the T2Cu center.B etween 218 and 238 K, the signal associated with oxidized nitrite-bound T2Cu "grows in" once again ( Figure 3B,S2and S8). We suggest that this increase in EPR signal associated with the T2Cu center is indicative of nitrite binding to the T2Cu after reduction of nitrite to nitric oxide,a nd subsequent nitric oxide dissociation.
Oxidized (Cu II )T 1a nd T2Cu signals of the "nitritebound", cryolytically reduced RpNiR core protein are presented in Figure 3D,F igure S3 (absolute EPR signal) and S9 (relative EPR signal). Between 150-180 K, areduction of both the T1 and T2Cu signals is observed, ar esult that suggests inter-copper electron transfer in the RpNiR core protein occurs in the absence of protein motions.A bove 180 K, there is an inverse trend in the signals attributed to the oxidized T1 and T2Cu centers ( Figure S3 and S9). From 180-210 K, the intensity of the oxidized T1Cu decreases,while the T2Cu II signal increases.H owever,b etween 210-270 K, ad ecrease in the oxidized T2Cu signal and an increase in the population of oxidized T1Cu are observed. Based on these data, we propose the following mechanism for the RpNiR core protein. In the oxidized form of the enzyme,20% of the T2Cu centers are occupied with nitrite.T he reducing conditions available for both T1Cu and T2Cu centers lead to the depopulation of the resting state EPR signal of the T2Cu center. This reduction of the T1Cu site appears to support binding of the nitrite substrate at the T2Cu site (shown by as hift in electronic properties and an increase in the Cu II signal at the T2Cu site). Following this,e lectrons transfer from the T1Cu to the T2Cu site,which is now fully occupied with nitrite.
In recent work, we proposed that electron delivery to the tethered RpNiR heme cofactor causes conformational change that is required for nitrite binding and catalysis in 3-domain NiRs. [15] We have also emphasized differences in catalytic mechanism between the full-length and core RpNiR proteins, which highlight previously unforeseen effects of tethering on enzyme catalysis.H ere,w es how that in the absence of the heme domain, the T1Cu of the RpNiR core protein must be partially or fully reduced to enable nitrite binding and catalysis,w hich provides new insight into the mechanisms of 2-domain NiRs.F or example,i thasb een shown that values for steady-state Michaelis constants (approx. 10 mm) [17] during catalysis and dissociation constants (approx. 350 mm) [33] for oxidized 2-domain NiR-nitrite complexes differ significantly. We have used a2 -domain NiR that (in the oxidized state) is only partially occupied with nitrite prior to performing cryoreduction-EPR. Our studies have shown that T1Cu reduction stimulates nitrite binding to the catalytic T2Cu center. This result likely accounts for the disparity in values of the Michaelis constants and NiR-nitrate complex dissociation constants noted above.

Conclusion
In summary,the cryoreduction-EPR method can be used to track inter-copper electron transfer in the copper nitrite . T1 to T2Cu electron transfer monitored in "nitrite-bound" AxNiR and RpNiR core proteins through the cryoreduction-EPR method. A) EPR spectra of oxidized "nitrite-free" (black, solid line) and "nitrite-bound" (red, solid line) AxNiR. B) EPR spectral changes showing T1 to T2Cu electron transfer when "nitrite-bound" AxNiR samples were annealed. C) EPR spectra of oxidized "nitrite-free" (black, solid line) and "nitrite-bound" (red, solid line) RpNiR core. D) EPR spectral changes showing T1 to T2Cu electron transfer when "nitrite-bound" RpNiR samples were annealed. All EPR measurements were performed at 20 K. reductase family of enzymes.O ur approach highlights the importance of protein dynamics in inter-copper electron transfer catalyzed by two-domain copper nitrite reductases. To the best of our knowledge,t his is the first spectroscopic study that has enabled direct monitoring of electron delivery, nitrite-binding and nitric oxide production at the T2Cu in this family of enzymes.O ur work has also enabled simultaneous observation of electron transfer between the T1 and T2 coppers in CuNiRs.T he approach we have developed is general and could be used to further understand intra-protein electron transfer in other multi-center copper-containing enzymes that have aminimal UV/Vis optical signal associated with the T2Cu sites such as laccases, [34] peptidylglycine aamidating monooxygenases [35] and particulate methane monooxygenases. [36]