Modulating the nitrite reductase activity of globins by varying the heme substituents: Utilizing myoglobin as a model system

    Modulating the nitrite reductase activity of globins by varying the heme substituents: Utilizing myoglobin as a model system Mary Grace I. Galinato, Robert S. Fogle III, Amanda Stetz, Jhenny F. Galan PII: DOI: Reference:

S0162-0134(15)30100-8 doi: 10.1016/j.jinorgbio.2015.10.010 JIB 9825

To appear in:

Journal of Inorganic Biochemistry

Received date: Revised date: Accepted date:

17 June 2015 8 October 2015 19 October 2015

Please cite this article as: Mary Grace I. Galinato, Robert S. Fogle III, Amanda Stetz, Jhenny F. Galan, Modulating the nitrite reductase activity of globins by varying the heme substituents: Utilizing myoglobin as a model system, Journal of Inorganic Biochemistry (2015), doi: 10.1016/j.jinorgbio.2015.10.010

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ACCEPTED MANUSCRIPT Modulating the Nitrite Reductase Activity of Globins by Varying the Heme Substituents: Utilizing Myoglobin as a Model System

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Mary Grace I. Galinato,a,* Robert S. Fogle III,a Amanda Stetz,a Jhenny F. Galanb,* a

School of Science-Chemistry, Penn State Erie, The Behrend College, Erie, PA 16563 Dept. of Marine Sciences, Texas A&M at Galveston, 200 Seawolf Parkway, Galveston, Texas 77553

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*Corresponding Authors: (M. G. I. Galinato) Email: [email protected]; Phone: 814-898-6004 (J. F. Galan) Email: [email protected]; Phone: 409-741-4326

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b

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ABSTRACT

Globins, such as hemoglobin (Hb) and myoglobin (Mb), have gained attention for their ability to reduce nitrite (NO2-) to nitric oxide (NO). The molecular interactions that regulate this chemistry

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are not fully elucidated, therefore we address this issue by investigating one part of the active site that may control this reaction. Here, the effects of the 2,4-heme substituents on the nitrite

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reductase (NiR) reaction, and on the structures and energies of the ferrous nitrite intermediates,

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are investigated using Mb as a model system. This is accomplished by studying Mbs with hemes that have different 2,4–R groups, namely diacetyldeuteroMb (-acetyl), protoMb (wild-type (wt)

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Mb, -vinyl), deuteroMb (-H), and mesoMb (-ethyl). While trends on the natural charge on Fe and O-atom of bound nitrite are observed among the series of Mbs, the FeII-NPyr (Pyr = pyrrole) and FeII-NHis93 (His = histidine) bond lengths do not significantly change. Kinetic analysis shows increasing NiR activity as follows: diacetyldeuteroMb < wt Mb < deuteroMb < mesoMb. Nitrite binding energy calculations of the different MbII-nitrite conformations demonstrate the N-bound complexes to be more stable than the O-bound complexes for all the different types of heme structures, with diacetyldeuteroMb having the greatest nitrite binding affinity. Spectral deconvolution on the final product generated from the reaction between MbII and NO2- for the reconstituted Mbs indicate the formation of 1:1 MbIII and MbII-NO. The electronic changes induced by the –R groups on the 2,4-positions do not alter the stoichiometric ratio of the products, resembling wt Mb.

KEYWORDS: Myoglobin, Heme, Nitrite, Reduction, Kinetics, Density Functional Theory

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ACCEPTED MANUSCRIPT 1. Introduction Nitrite has emerged to be a significant source of nitric oxide (NO) in blood and tissue, with as much as 70% of plasma nitrite originating from endothelium nitric oxide synthase [1]. Under

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oxygen-limiting conditions, NO generated from nitrite (NO2-) is responsible for several signaling

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processes, including hypoxic vasodilation [2-4], cytoprotection after ischemia [5,6], and hypoxic mitochondrial function [7]. Although several enzymes/systems utilize unique pathways to

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generate NO from NO2- [8-12], members of the globin family ubiquitously catalyze the reduction of NO2- to NO [7,13-17], thus implicating an essential functionality in this class of enzyme. The

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reaction of nitrite with five-coordinate deoxyhemoglobin (deoxyHb, HbII) has been investigated in the past several decades [13,14], but is still generating interest because of the physiologic

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effects it displays in vivo [18,19]. However, given the complex kinetics of NO2- reduction as reflected by the different bimolecular rate constants in the R- and T-states of Hb [20-22], a simpler globin such as myoglobin (Mb) would be a more ideal system to investigate.

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Interestingly, Mb also shows nitrite reductase (NiR) activity, as demonstrated by its ability to

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modulate mitochondrial respiration in isolated mitochondria, cardiomyocytes, and heart homogenates [7]; and reduce myocardial infarction and improve recovery of post-ischemia in

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mice [19]. During the catalytic process, the following reactions occur [7]: MbII + NO2- + H+ MbII + NO

k2

k1

MbIII + NO + OH-

MbII-NO

(1) (2)

Here, reaction (1) is rate-limiting, presumably proceeding through the formation of a MbIII-NO intermediate, where the MbIII-NO (a six-coordinate ferric complex) bond is labile and prone to dissociation [23,24]. The second reaction occurs very rapidly due to the high binding affinity and slow dissociation constant of NO to MbII [25]. The products MbII-NO and MbIII are produced in 1:1 stoichiometric amounts [7], which are also supported by our results (see discussion). Although the detailed mechanism of NO2- reduction to NO in globins is still unknown, the Mb NiR reaction may be postulated to occur via different mechanisms, as described in ref. [26] and presented in Scheme 1. In the first mechanism, nitrite binds to FeII and is stabilized through a hydrogen bond with His64 (Scheme 1, route 1). In the presence of protons, a MbIII-NO/MbIINO+ intermediate forms. The labile nature of the MbIII-NO bond generates MbIII and free NO. Nitric oxide then rapidly binds to free MbII, forming the stable MbII-NO product. Alternatively, 2

ACCEPTED MANUSCRIPT nitrous acid may be the bound ligand to the Fe center (Scheme 1, route 2) [14,26]. In both cases, nitrite binds to FeII through nitrogen (N-bound, nitro) similar to that observed in cd1 nitrite reductase [27]. With respect to the identity of the ligand binding to the Fe center (e.g. nitrite or

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nitrous acid), the experimental pH has to be considered. Nitrous acid is fairly weak (pKa = 3.15) and nitrite dominates in solution under the pH we use for our studies, pH 7.4, (ratio of

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[HNO2]/[NO2-] is ~10-5), hence, we focus only on the nitrite ligand in our work. Lastly, another

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potential route towards the generation of NO involves nitrite being O-bound to the Fe center (Scheme 1, route 3). The formation of this linkage isomer is supported by a crystal structure of a

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MbIII-NO2- complex showing nitrite O-bound (nitrito) [28] (when exposed to high intensity synchrotron X-ray radiation, this ferric nitrite complex can be reduced while retaining its

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conformation [29]). More recently, evidence from quantum (QM) calculations and EPR measurements demonstrate that nitrite binds to ferric heme via the O-atom in a type-II center [30]. While any of these mechanisms are conceivable, the possibility of both isomers being

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present in solution during the NiR reaction cannot be discounted. It has been shown that both N-

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and O-bound MbIII-nitrite linkage isomers may simultaneously be present in solution [31]. This implies that the ferrous MbII-nitrite complex may also potentially exist as two isomers in

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solution.

Among the members of the five-coordinate globin family, Mb is the most robust and has been the most structurally-characterized, making it an ideal system to test for the active site components that regulate the NiR chemistry, and serve as a model for Hb. Although the overall process of NO generation through NO2- reduction has been examined, detail on its mechanism is still lacking. However, studies that provide more insight into this chemistry have emerged. For example, the distal pocket residues affect the NiR activity of MbII as demonstrated by a change in the nitrite reduction kinetics of the Mb mutants, H64V and H64V/V67R, relative to wild-type (wt) [32]. The distal residue His64 forms a crucial H-bond that influences the mode of ligand coordination on the Fe center. Due to the nature of the short-lived intermediate, the mode of nitrite coordination to MbII is unknown. However, crystal structures of the ferric complexes of wt Mb and Mb mutants show that nitrite interconverts between the nitrito and nitro conformation in the presence (H64 and H64V/V67R) and absence (H64V) of the H-bond, respectively [32]. Another component that influences the kinetics of nitrite reduction is the metal center. Mn II- and CoII-substituted Mbs generate functional enzymes whose NiR activities are lower than wt [33]. 3

ACCEPTED MANUSCRIPT The difference in activity is attributed to the strength of NO2- binding to the metal center as influenced by its electronic configuration. Lastly, the redox potential is shown to affect NiR activity of globins, as demonstrated by the rate of NO2- reduction in Mb (46 mV vs SHE) being

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36x faster than that of Hb (139 mV vs SHE) [7].

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Beyond the above-mentioned components, other factors that may influence NO2- to NO conversion in Mb have yet to be explored. The goals of this work are to elucidate the relationship

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between the Mb NiR activity and the electronic properties of the heme peripheral side chains; and probe the structure of the MbII-nitrite intermediate complex and nitrite binding energies as a

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function of the heme substituents. In order to achieve these goals, Mb samples having hemes with altered peripheral –R groups are explored via kinetic experiments, and structure and

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energetic calculations. For globins [34,35] and cytochrome P450s [36], their ligand-binding properties and catalytic activities are shown to be strongly affected by modifying the substituents at the 2- and 4-positions instead of the 6- and 7-positions for which horseradish peroxidase [35]

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is more sensitive to. For Mb in particular, the altered ligand affinities are thought to be a

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consequence of site-specific protein side-chain interactions [37]. In this current study, we focus on a set of hemes in which the 2,4–R groups are different relative to wt. Although the effect of

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heme modification on the O2, CO, N3-, and CN- binding affinity in Mb has been investigated [34,35], its influence on the NiR activity of Mb has not yet been examined. We systematically investigate reconstituted Mb samples that contain the following hemes: diacetyldeuteroheme, protoheme, deuteroheme, and mesoheme (Figure 1), which have acetyl, vinyl, hydrogen, and ethyl groups at the 2,4-positions, respectively. This provides a set of Mbs with heme –R groups whose electron-releasing capacity increases in the order stated. The influence of the heme substituents on its electronic structure is manifested through the charge on the iron center, and correspondingly, its redox potential. Within the series of Mbs investigated in this work, the reduction potential decreases in the following order: wt Mb (-140 mV) > deuteroMb (-162 mV) > mesoMb (-170 mV) (reference electrode is Ag/AgCl) [38], illustrating the electronic effect of the 2,4-heme R groups. In addition to probing the slow reaction of MbII with nitrite, we also use density functional theory (DFT) calculations to investigate the structure of MbII-nitrite intermediate complexes and nitrite binding affinities as influenced by the heme substituents at the 2,4-positions. This part of the study utilizes model systems generated based on the nitrite-

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ACCEPTED MANUSCRIPT bound myoglobin crystal structure with the heme in the ferric form [28]. Other possible nitrite binding modes [39] were also explored for the Mb systems.

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2. Materials and Methods 2.1. Reconstitution and purification of Mb samples

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FeIII 2,4-diacetyldeuteroporphyrin IX chloride, FeIII mesoporphyrin IX chloride, and FeIII

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deuteroporphyrin IX chloride were purchased from Frontier scientific at 95% purity. Horse heart (hh) wt Mb was purchased from Sigma-Aldrich in the pure metMb (FeIII) form. In order to

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generate the reconstituted Mb samples (diacetyldeuteroMb, mesoMb, and deuteroMb), a twostep procedure involving apoMb preparation and modified-heme reinsertion was employed

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according to Teale methodology [40]. For each reconstituted Mb preparation, 10 mg of hh wt Mb crystals was dissolved in 5 mL deionized water. The heme was extracted by decreasing the pH of the solution to 1.5, after which it was separated from the apoprotein by adding 20 mL of 2-

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butanone following extraction. The apoprotein was dialyzed overnight against deionized water

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followed by phosphate buffer, pH 7.4. The solid porphyrin was dissolved in minimal amounts of dimethylsulfoxide and mixed into an ice-cold apoMb solution at a molar ratio of 1.5:1 (heme:

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apoMb). The reconstitution progress was monitored by absorption spectral changes. The mixture was further dialyzed against 100 mM phosphate buffer pH 7.4, and concentrated to a volume of ~0.5 mL. All reconstituted and wt Mb samples were then purified through ion-exchange (CM-52 cellulose, Serva) followed by gel-filtration (Sephadex G-25, Sigma Aldrich) columns that were pre-equilibrated with phosphate buffer, pH 7.4. After purification, only the enzymes that had a high ratio of the Soret-to-protein bands (RZ values) were used: diacetyldeuteroMb (RZ > 2.4); wt Mb (RZ > 5); deuteroMb (RZ > 4.6); mesoMb (RZ > 4.6). The extinction coefficients of the reconstituted samples were obtained through the pyridine haemochromogen method [41] and comparison with prior studies [35]. All samples were stored in a -80 C freezer. It has been previously reported that hemes are able to insert into the apoprotein either in a “normal” or “rotated” (180 about the - axis) orientation and are formed in equal amounts upon reconstitution, thus creating heme orientational disorder [34,42]. The substituents in the 2,4-position particularly influences the rate of heme reorientation [43]. However, in this activated process (21 kcal/mol for wt Mb), the heme eventually reorients and equilibrates to the more stable orientation [37,44]. This is particularly true for meso- and diacetyldeuteroMbs, 5

ACCEPTED MANUSCRIPT which when compared to native Mb show similar exchange rates of key protons arising from His residues [45], indicating that the reconstituted enzymes have the same heme cavity structure as native Mb. Interestingly, a more recent study demonstrates orientational heme disorder in

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mesoMb [34], the disparity with ref [45] of which may originate from slightly different methods of preparation and equilibration time. In our work, we allow the reconstituted Mbs to settle at

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4C for a few hours before storing right after reconstitution, and at 20 C for one hour prior to

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kinetic analyses, allowing the heme to equilibrate to the normal orientation. At this temperature and duration of equilibration, the heme redistributes to the 90/10 equilibrium ratio and the

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normal orientation predominates, with minor amounts of a different conformer [44]. For deuteroMb, which is devoid of bulky substituents at the 2,4-positions that stabilize the heme

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within the pocket, heme disorder is apparent [42]. Therefore, the reactivity of deuteroMb may also be influenced by the presence of multiple conformers in solution, and experimental data obtained from this system represents an average effect of the different conformers.

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In order to generate reference absorption spectra, the MbII-NO and MbIII-NO2- forms of all

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holoenzymes were obtained using the following procedure. In producing MbII-NO, nitric oxide gas (> 99%, Praxair) was scrubbed through KOH pellets to remove trace quantities of impurities.

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The gas was bubbled into a previously freeze-pump-thawed solution of 100 mM phosphate buffer pH 7.4, for several minutes. The solubility of NO gas in buffer is ~1.7-2 mM [46]. Based on its solubility, stoichiometric amounts of NO were added into a MbII solution having a known concentration. Pure MbII was prepared in a glovebox as described below. For MbIII-NO2-, stoichiometric amounts of standard 100 mM nitrite solution (Sigma Aldrich) were added to a known concentration of MbIII (~10 M). The weak binding of nitrite to MbIII (Knitrite = 120 M-1 for wt Mb) [33] was accounted for during the reaction. In generating the ferric complex of the reconstituted Mbs, the Knitrite of wt Mb was used as an estimate in determining the stoichiometric amounts required of the reactants to achieve the formation of the complex. In order to determine the nitrite binding constant of the Mbs studied, a solution of Mb was titrated with known amounts of nitrite while monitoring the spectral changes in the 450-700 nm region [33]. Knitrite was extrapolated from the sigmoidal data generated from plotting the log (Abs) as a function of log [NO2-]. All Mb samples were kept in 100 mM phosphate buffer, pH 7.4. 2.2. Kinetics experiments on Mb samples 6

ACCEPTED MANUSCRIPT MbII solutions were prepared in a glove box (100% N2 gas, Coy Laboratories) by adding sodium dithionite to a MbIII solution that has previously been stored in deoxygenated 100 mM phosphate buffer pH 7.4. The reducing agent was removed by passing the sample through a

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Sephadex G-25 column pre-equilibrated with O2-free phosphate buffer pH 7.4. It was ensured that the pH remained constant at 7.4 for all samples, since [H+] affects the kinetics of the reaction

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between MbII and nitrite [7]. Each MbII solution (600 L) was sealed in an airtight cuvette

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(Starna) containing a microstir bar. The purity of the solution was verified and confirmed using a Red Tide UV-vis spectrometer (Ocean Optics) housed in the glovebox.

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An anaerobic standard solution of nitrite (100 mM, Sigma Aldrich) was used as a stock solution, and aliquots were added to the cuvette using a Hamilton syringe to obtain the

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experimental nitrite concentrations. A custom-made sample cuvette holder containing a magnetic stir plate was utilized to ensure that the reactants were properly mixing during the course of the reaction.

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Kinetic traces were obtained by monitoring the decrease of the MbII Soret band (448 nm for

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diacetyldeuteroMb, 434 nm for wt Mb, and 421 nm for both deutero- and mesoMbs) using a Cary 50 spectrophotometer (Agilent). Single exponential fits of the traces were conducted using

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the built-in software. The observed rate constant at each nitrite concentration was plotted, employing a linear fit to generate the bimolecular rate constant. The absorption spectra of the final product from the reduction reaction was acquired and analyzed for contributions originating from MbII, MbII-NO, MbIII, and MbIII-NO2- by fitting the final spectra to the reference spectra of these components. Analyses of the linear plots and the end-product absorption spectra were conducted using Origin v.9.0.0 (OriginLab Corporation). 2.3. Density functional theory (DFT) calculations Structural models for the reconstituted Mb active sites with varying heme structures were generated based on the high-resolution crystal structure of MbIII-NO2- complex (PDB code: 2FRI) [28]. We focused on nitrite (instead of nitrous acid) as the ligand bound to MbII in order to simulate the most likely form of the ligand under our experimental conditions (e.g. at pH 7.4, the ratio of [HNO2]/[NO2-] is ~10-5 in aqueous solution). For the MbII-NO2- complexes, the models consist of the heme, nitrite bound to iron, and the proximal and distal His ligands. The His ligands were simplified by truncating the carboxyl and amide heavy atoms with only the alpha 7

ACCEPTED MANUSCRIPT carbons (Cα) and the His side chain included in the calculations. The heme structures were generated by modifying the –R groups at the 2- and 4-positions from vinyl (wt Mb) to acetyl, hydrogen, and ethyl for diacetyldeuteroMb, deuteroMb, and mesoMb, respectively. Since the

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conformation of the MbII-NO2- complex is unknown for the reconstituted Mbs, four possible conformations were tested (Figure 2), one based on the FeII-NO2- complex in cd1 bacterial nitrite

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(N-bound) reductase [27], and three based on the FeIII-ONO- structure of Hb (O-bound) [47]. All

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atoms of the active site were allowed to optimize while the Cα of both His residues were kept frozen. This allows the proximal FeII-NHis93 bond, distal FeII-nitrite bond and the H-bond

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between distal His and nitrite to optimize based on the electron densities as influenced by the –R groups at the 2,4-positions. Geometry optimizations were carried out using the BP86 functional

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[48,49] and LanL2DZ* basis set, which uses polarization functions from TZVP on all nonhydrogen functions [50] applied to LanL2DZ basis set [51-53]. To our knowledge, no one has been able to experimentally verify the spin state of the elusive globin ferrous nitrite complex.

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However, on the basis of nitrite being a strong field ligand, the spin state of the Fe II center can

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presumably be assigned as S = 0. For model systems with unbound nitrite, calculations using both S = 0 and S = 2 spin states were carried out and energies were compared. Experimentally,

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the penta-coordinated unbound complex is known to be high spin [54-56], however, theoretical calculations also showed that the spin state is ambiguous [57]. Using the optimized geometries, single point energies for each complex were calculated at the BP86/TZVP level of theory with Grimme’s D3 dispersion model [58]. TZVP applies the Ahlrich’s triple- valence polarization basis set [59], while LanL2DZ utilizes the Dunning/Huzinaga full double- (D95) basis functions on first row atoms and Los Alamos effective core potential plus DZ functions on other elements [52,60-62]. In determining the nitrite binding energy, the geometries and energies of the heme active site in the presence and absence of the NO2- ligand were calculated. NPA charge and NBO calculations [63] were carried out using NBO 3.1 within the Gaussian 09 program [64,65]. All geometry optimizations and energy calculations were carried out using Gaussian 09 [64]. Natural bond orbitals were plotted using the Gaussview v.5 [66] program. 3. Results 3.1. UV-visible absorption spectra 8

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The absorption spectra of MbIII, MbII, MbII-NO and MbIII-NO2- for all the systems studied are shown in Figures 3 and S1. In general, the overall spectral shapes of the bands in the Soret and

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Q-regions of wt Mb, deuteroMb, and mesoMb are fairly similar, however, their relative intensities vary. In addition, the peaks in wt Mb are shifted to even lower energy by ~10-15 nm

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relative to those of deuteroMb and mesoMb. Among the Mb samples investigated, the absorption spectra of diacetyldeuteroMb are shifted to lower energy by approximately 10-15 nm relative to

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the bands in wt Mb, and also display lesser resolved features. For example, while a two-peak spectral feature (with distinct bands or noticeable shoulders) is observed in the Q-region of the

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MbII-NO complex for wt Mb, deuteroMb, and mesoMb, a broad band with a subtle shoulder is apparent in diacetyldeuteroMb. The presence of acetyl groups in the 2,4-positions of the heme

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(as in the case of diacetyldeuteroMb) broadens the spectral features in the Soret region as demonstrated by an increase in the full-width at half maximum. The broad Soret band, and the

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weaker absorbance features of diacetyldeuteroMb relative to the other three Mbs studied in this

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work, are consistent with previous studies [37,67]. Given the presence of heme orientational isomers in deuteroMb [42,43], it is possible that the spectral profile of its different forms (e.g.

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MbIII, MbII, MbII-NO and MbIII-NO2-) is a reflection of a mixture of the isomers. As the hemes in diacetyldeuteroMb and mesoMb have the dominant normal conformer oriented in the protein pocket but potentially with minor contributions of the reversed orientational isomer [34,37], the presence of the two isomers in solution for both Mbs is also conceivable. The slow reaction between MbII and NO2- was monitored for wt and the reconstituted Mbs using steady-state absorption. In all samples, isosbestic points are observed during the transition from MbII to the final product (Figure 4). The characteristic Soret band of the MbII form is shifted to higher energy and the Q bands appear distinctly unique upon reaction with nitrite, indicative of NO2- to NO conversion (Figure 4) [17]. For example, the Soret band of diacetyldeuteroMb (448 nm), wt Mb (434 nm), deuteroMb (421 nm), and mesoMb (421 nm) shifts to 431, 412, 397, and 396 nm, respectively. In addition, new features in the Q-region emerge from the product formed between the reaction of MbII with NO2-, which are particularly more apparent for wt Mb, deuteroMb, and mesoMb. Here, the single band at 554 nm (wt), 543 nm (deuteroMb), and 544 nm (mesoMb) changes into a four-peak feature at 503, 547, 583, and 631 nm (wt); 496, 529, 568, and 623 nm (deuteroMb); and 490, 532, 569, and 624 nm 9

ACCEPTED MANUSCRIPT (mesoMb). In diacetyldeuteroMb, the single band at 561 nm converts to a band at 552 nm and a subtle shoulder at 588 nm. Sample kinetics traces of these changes are presented in Figure S2. According to equations (1) and (2), a 1:1 ratio of MbIII and MbII-NO is produced when

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stoichiometric amounts of MbII and NO2- react. The end-point absorption spectrum obtained from this reaction is analyzed for the contributions of its components by fitting it to a sum of the

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reference spectra of MbIII, MbII-NO, and MbII (Figure 5). For deutero- and mesoMb, the ratio is

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50% MbIII and 50% MbII-NO. In the case of diacetyldeutero- and wt Mb, the final spectra is best fit with minor amounts of MbII (2-4%) and approximately equal amounts of MbIII and MbII-NO

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(47-49%). The stoichiometric ratio of the components that comprise the final product are consistent with previous studies on wt Mb [7] as well as other members of the globin family such as human hemoglobin (Hb) [17] and Synechocystis Hb [15]. The implications on the formation

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of stoichiometric MbIII and MbII-NO on the mechanism of NO generation are discussed below.

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3.2. Slow kinetics data

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Nitrite reduction by wt MbII and several wt HbsII have been previously investigated and can be described by a two-step process as shown in equations (1) and (2) [7,15]. Reaction 1 is rate-

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controlling and most likely proceeds through the formation of MbIII-NO followed by NO release (14 s-1) [68]. Reaction 2 is rapid, with a high bimolecular rate constant of NO binding to MbII (1.7 × 107 M-1 s-1) and a low dissociation rate constant (1.2 × 10-4 s-1) [69]. At constant pH, the overall rate of reaction can be quantified by monitoring the disappearance of Mb II according to the following relation:

In excess nitrite concentrations, the rate law can be simplified to a pseudo-first order reaction where a linear fit to a plot of kobs versus [NO2-] generates the bimolecular rate constant. Figure 6 shows the observed reaction rates for each Mb sample as a function of nitrite concentration. At 20 C and pH 7.4, the bimolecular rate constant increases in the following order: diacetyldeuteroMb (1.2 + 0.1 M-1 s-1) < wt Mb (2.2 + 0.2 M-1 s-1) < deuteroMb (4.9 + 0.3 M-1 s-1) < mesoMb (7.4 + 0.4 M-1 s-1). The rate constant for wt Mb increases from 6 to 12 M-1 s-1 at 25 C and 37 C, respectively [7], which is an expected trend when temperature rises within the range that an enzyme can tolerate without yet affecting function. 10

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3.3 Computational analyses of NO2- binding: structure, binding modes, and energies To further elucidate the effects of altering the 2,4-heme substituents on globin NiR activity, a

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series of DFT calculations were performed to examine the structure, NO2- binding modes and energetics of NO2- complexation to FeII for the different Mbs studied in this work. This section

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first focuses on the geometric structure of our model MbII systems, followed by that of the fast-

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forming intermediate, MbII-nitrite. While our experimental results do not probe the formation or loss of the MbII -nitrite intermediate, the computational data generated provides another level of

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understanding into the ligand binding affinities, structure and energies of the MbII-nitrite linkage isomers as a function of heme substituents. It is noted that the computational discussions

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pertaining to unbound MbII is based on S = 2 spin state calculations, as penta-coordinated ferrous Mb is known to display a quintet spin state [54-56]. However, we also calculated geometric structures for the S = 0 spin state as seen in Table 1.

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3.3.1. DFT calculations of MbII with modified heme structures Modifying the substituents at the 2,4-heme positions within the series of Mb model systems

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results in negligible differences on the average heme pyrrole nitrogen distance (FeII-NPyr). The various substituents induce the following FeII-NPyr distances: acetyl (diacetyldeuteroMb), 2.092 Å; vinyl (wt Mb), 2.089 Å; H (deuteroMb), 2.092 Å; ethyl (mesoMb), 2.079 Å. Although the differences are very minor, the electron withdrawing substituents (as in the case of diacetyldeuteroMb) tend to cause a decrease in electron density on the heme resulting in a longer FeII-NPyr average distance. Our computational results are in agreement with a previous X-ray absorption study on sperm whale Mbs reconstituted with the same set of hemes investigated in our work [70], where the FeII-NPyr average distances yielded insignificant differences among the various hemes. In addition, varying the peripheral –R groups in these positions only very slightly modulates the proximal FeII-NHis93 bond. The differences in the FeII-NHis93 distance among the various heme types are minute, with deuteroMb and wt Mb having the longest and shortest FeIINHis93 distance at 2.107 Å and 2.095 Å, respectively. Like that observed for the FeII-NPyr average distance, the trends in the FeII-NHis93 bond length are consistent with a previous work [70]. In their work, the authors demonstrate that the proximal Fe-NHis93 bond length of the ferric form of the Mb samples remains unchanged within error. This is consistent with the trends we observe 11

ACCEPTED MANUSCRIPT for ferrous Mb, which shows only a 0.005 Å standard deviation in the Fe-NHis93 bond distance among all samples investigated. A complete list of the geometric parameters is given in Table 1. In order to quantitatively illustrate the effect of the 2,4-R groups on the heme electronic

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structure, natural population analyses (NPA) [63] were carried out. Introduction of acetyl, ethyl, hydrogen, and vinyl groups changes the natural charge on the Fe center in the following order:

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diacetyldeuteroMb (0.880) > deuteroMb (0.872) > wt Mb (0.871) > mesoMb (0.858). The acetyl

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group in diacetyldeuteroMb withdraws electron density from the porphyrin system, therefore increasing the natural charge of Fe. The trends in the electron distribution for each Mb as a

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function of the –R groups on the 2,4-heme positions is presented in Figure S3. The 2,4-heme substituents can influence the heme electronic structure in two ways: (1) inductive effects, and (2) resonance effects. For example, the electronegative nature of the carbonyl oxygen in acetyl

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tends to draw electron density away (inductive); while the lone pair -orbitals on the same oxygen atom is involved in the -conjugated network with the porphyrin ring (resonance). In the

D

case of wt Mb, the electron withdrawing nature of the vinyl group pulls electron density away,

TE

hence a relatively high partial charge on the Fe center is observed. However, it is slightly lower in comparison to diacetyldeuteroMb since the oxygen atom in the acetyl group of this

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holoenzyme is more electronegative. Compared to diacetyldeuteroMb and wt Mb, addition of an ethyl group as in the case of mesoMb increases the electron density on the porphyrin, thereby decreasing the natural charge of Fe. 3.3.2. Geometric and electronic structures, and energies of MbII-nitrite complexes In this section, we first look at the effect of varying the 2,4-substituents on the heme geometric structure of the MbII-nitrite intermediate. For each Mb sample, the change in the average FeII-NPyr distances (2.027-2.042 Å) is insignificant in the presence of bound nitrite. However, a very slight increase in the FeII-NHis93 bond lengths is observed for the N-bound complexes over the O-bound forms. Within the series of Mb samples, the average FeII-NPyr and FeII-NHis93 bond lengths do not significantly differ among one another. Here, the standard deviation of the FeII-NPyr bond lengths are ~0.004 (N-bound) and 0.007 (O-bound) Å; while that of the FeII-NHis93 bond distances are ~0.020 (N-bound) and ~0.009 (O-bound) Å. Beyond the electronic influence of nitrite on the heme environment, we probe the effect of the heme electronic structure on the energy of the MbII-nitrite intermediate complex. During the 12

ACCEPTED MANUSCRIPT NiR process, MbII reduces NO2- to NO via a mechanism similar to cd1 nitrite reductase, where a MbII-nitrite intermediate forms prior to NO generation. Crystal structures of the ferrous and ferric forms of Mb-nitrite show an O-bound ligand (nitrito) [28,29], as opposed to the FeII-NO2-

PT

complex of cd1 nitrite reductase [27] where nitrite is N-bound (nitro). The preferred isomer (Nbound or O-bound) of the nitrite complex can be hypothesized to influence the reaction pathway,

RI

as shown in Scheme 1. From our DFT calculations on the series of Mb model compounds, the N-

SC

bound complexes are predicted to have the lowest binding energies (Table 2). Previous DFT studies of the corresponding ferric forms of the complexes with distal His64 incorporated in

NU

calculations also shows the N-bound complex to be more stable [30,39]. Based on our model systems, His64 plays a role in stabilizing both forms of the MbII-nitrite complex through the N-

MA

H(His)…O(NO2-) and N-H(His)…N(NO2-) H-bonding interaction. Aside from His64, other protein residues can possibly interact with the ligand and enhance the stability of both conformers, thus lowering the energy difference between the N-bound and O-bound complexes.

D

The fact that the less stable O-bound nitrite linkage isomer has been observed in Mb [29] helps

TE

establish that the protein matrix (possibly beyond the immediate His residues) stabilizes the complex. Perturbation by the solvent may also contribute to this behavior. Despite our

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calculations showing that the nitro complexes are more stable by ~5.0 kcal/mole (average Nbound versus O-bound conf1 difference), the possibility of the nitrito conformer being present in solution during the NiR reaction cannot be discounted (see discussion below). In order to assess the stability of the bound nitrite, the FeII-NO2- bond lengths were calculated, with particular focus on the N-bound and O-bound conformation 1 linkage isomers of the set of Mbs studied. Within the series, the FeII-NO2- bond length slightly increases in the following order: deuteroMb (1.939 Å) < diacetyldeuteroMb (1.942 Å) < wt Mb (1.947 Å) < mesoMb (1.952 Å). The addition of electron releasing groups on the 2,4-positions increases the FeII-NO2- bond length. A fairly similar trend is observed for the MbII-nitrito complexes (as seen for conformation 1), with the exception that diacetyldeuteroMb now has the smallest Fe-ONObond length: diacetyldeuteroMb (1.974 Å) < wt Mb (1.977) wt Mb and deuteroMb (1.980 Å) < mesoMb (1.984 Å). The FeII-nitrite bond lengths are comparable to those obtained previously for a model of cd1 NiR where nitrite is N-bound to the iron center (~1.90 Å) [71], as well as with the calculated FeIII-nitrite bond lengths in globins (~1.95 Å) where the peripheral heme side chains are truncated [30,39]. In general, the heme peripheral electron releasing substituents causes the 13

ACCEPTED MANUSCRIPT FeII-ONO- bond to lengthen, hence, destabilizing the nitrite complex. The Fe-ONO- bond lengths for ferrous wt Mb are also comparable with the ferric nitrito protein crystal structures of ~ 2.0 Å (PDB: 3LR7 [29] and 3D7O [47]), and with that obtained from a DFT globin model system

PT

where the peripheral side chains are truncated (1.900 Å) [39]. A complete list of the geometrical parameters is presented in Table 1.

RI

Natural Bond Orbital Analyses (NBO) of the various heme systems further support the

SC

observed trends in the Fe-nitrite bond strength as the substituents are varied. For illustration purposes, orbital analyses of N-bound complexes are discussed in this section. Figure 7 shows

NU

the relevant σFe-NO2 natural bond orbitals for the N-bound linkage isomers of the Mbs studied. For diacetyldeuteroMb, the Fe-NO2- σ orbital has 73.2 % N (NO2-) and 26.80 % Fe, while for

MA

mesoMb the contributions to the Fe-NO2- σ bond changes to 74.45% N (NO2-) and 25.55% Fe. A decrease in the iron contribution in mesoMb suggests a weaker interaction between the nbN-nitrite and the dz2 orbital of iron. Analysis of the occupancy of the 3dz2 natural atomic orbitals shows

D

the following trend: diacetyldeuteroMb (1.488) < wt Mb (1.557) < deuteroMb (1.600) < mesoMb

TE

(1.631), which illustrates the electronic effect of the peripheral substituent on the electron density of the iron center. Further, second order perturbation analyses show a large interaction between

AC CE P

the nbHis-93 N atom orbital and a Fe* acceptor orbital. Although the differences are relatively small, this type of stabilizing interaction increases with electron releasing peripheral substituents, and is most predominant for mesoMb. The large interaction energy for mesoMb indicates a strong FeHis93 interaction, ultimately causing a weakening of the FeII-nitrite bond trans to the His93 ligand. These results support the trends in Fe-NO2- bond lengths (see Table 1). In the case of Obound nitrite complexes with the exception of deuteroMb, natural atomic orbital analysis also shows a decrease in the occupancy of the 3dz2 orbitals with more electron withdrawing heme substituents, resulting in a lower electron density of the metal center. The trend is as follows: diacetyldeuteroMb (1.127) < deuteroMb (1.611) < wt Mb (1.722) < mesoMb (1.788). Similarly, the interaction of His93 with the iron center (nbHis-93 N atom orbital and a Fe* acceptor orbital) is smallest with diacetyldeuteroMb and largest with mesoMb, causing a trans effect in terms of the strength of the FeII-ONO- bond. The relative stabilities of the different N- and O-bound MbII-nitrite complexes were obtained by calculating the nitrite binding energies (ΔEbinding = [EMb(II)-nitrite]bound – [EMb(II) + Enitrite]unbound). Two sets of calculations with different spin state assignments were carried out for the unbound 14

ACCEPTED MANUSCRIPT complex, S = 0 and S = 2. Taking the case of S = 2, our results show that the relative binding energies of N-bound MbII-nitrite complex increases in the following order: diacetyldeuteroMb (0.00 kcal/mol) < wt Mb (1.12 kcal/mol) < mesoMb (2.40 kcal/mol) < deuteroMb (8.33

PT

kcal/mol). For conformation 1 of the O-bound MbII-nitrite complex, the binding energy increases as follows: diacetyldeuteroMb (0.00 kcal/mol) < wt Mb (3.87 kcal/mol) < mesoMb (5.23

RI

kcal/mol) < deuteroMb (7.96 kcal/mol). Similar trends were obtained for other O-bound

SC

conformations. Previous investigations on ferric Mb with nitrite bound through N- and O-atoms show that the nitro complex is the most stable [30,31,39]. Among the O-bound isomers studied

NU

in our work, the ΔEbinding for the linkage isomers where the O-atom of nitrite is oriented towards the distal His (conf1 and conf3) are more stable than when it is pointed away (conf2). This type of conformation allows for efficient H-bonding of the nitrite O-atom and the distal His. A

MA

complete list of energies for the different MbII-nitrite conformations explored in this work is

D

presented in Table 2.

TE

4. Discussion

4.1. Effect of 2,4-R groups on the heme environment

AC CE P

The substituents on the 2,4-heme positions induce geometric and electronic effects that are reflected in spectral changes, kinetics, and energetic calculations of the Mb samples investigated in this work. Modifying the peripheral heme 2,4-R groups into more electron releasing side chains increases the heme  electron density, and subsequently alters the charge on the Fe center. Upon complexation with nitrite, the electron density from the ligand is delocalized to the metal center, slightly decreasing the partial charge of Fe. As mentioned earlier, the FeII-NHis93 bond lengths vary within the Mb series (2.095 to 2.107 Å) but the distances are within error, consistent with a previous work [70]. Addition of nitrite to the Fe center increases the Fe II-NHis93 bond lengths for the nitro (2.118 to 2.146 Å) and nitrito isomers (~2.002 to ~2.050 Å across the three conformations studied) (Table 1). Importantly, the strength of the FeII-NHis93 bond relates to the ligand reacting trans to the His93 residue [72,73], and therefore may provide mechanistic insight into a catalytic reaction. However, in the Mb series investigated in this work, the FeII-NHis93 bond distances do not considerably vary across the different types of heme despite the somewhat substantial difference in NiR activity of each Mb sample. This suggests that the Fe II-NHis93 bond

15

ACCEPTED MANUSCRIPT strength induced by the 2,4-heme substituents studied in this work has minimal influence on the NiR functionality of Mb and globins in general. The protein matrix also plays an important role in modulating the geometric and electronic

PT

properties and/or orientation of the heme by inducing a global change on the heme environment, effectively altering its electronic properties and/or orientation. In hh Mb [74], the protein

RI

environment at the 2-position is different from that of the 4-position (Figure S4). For example,

SC

only a few protein residues are within the vicinity of the 2-heme position, which include a distant hydrophobic cluster consisting of Phe138, Ile111, and Leu72. On the other hand, the 4-heme

NU

position has Phe43, Leu32, Leu104 and polar Thr39 residing nearby. A previous resonance Raman study on whale Mb reconstituted with different hemes, including deutero- and 2,4diacetyldeuterohemes which are used in our work, shows that the protein influences the

MA

porphyrin -system primarily through the 2-substituent [37]. Based on the binding site models generated for the different types of Mbs, Phe138 is within ~3.3-3.8 Å of the substituent at the 2-

D

position (measured between closest heavy atom between substituent and Phe; this distance is not

TE

representative of that in deuteroMb, which has a –H substituent). This suggests that the substituent at this position may be affected by long-range dispersive interactions rather than

AC CE P

steric effects (Figure S4). A similar effect would be expected at the 4-position, where the Phe43 side chain is within ~3.5-3.9 Å of the substituent. Secondary electronic effects are also possible in the presence of a polar Lys45 residue, which is within the vicinity of Phe43. Importantly, the interaction of these residues with the heme varies with the nature of the substituent and geometry of the pocket. For example, the interactions of the polar acetyl substituents and nearby residues are different compared to the alkyl substituents. Further, in the case of deuteroheme, the small size of the –H substituent causes greater distortion in the binding pocket, thus allowing more flexibility for heme rotation in the holoenzyme. This effect is reflected in the stability of the heme within the protein pocket of deuteroMb. Given the bulky substituents at the 2,4-positions of diacetyldeuteroMb (acetyl) and mesoMb (ethyl), these –R groups are able to stabilize the heme such that it exists in one predominant normal orientation [45], although minor amounts of the reverse heme orientational isomer may still be present. On the other hand, the –H groups on deuteroMb are too small to interact with the residues, therefore providing the heme with more flexibility within the active site pocket, resulting in greater orientational disorder [42,43]. This in turn may affect its NiR activity. 16

ACCEPTED MANUSCRIPT

4.2. Correlation between the heme structure and the NiR activity of Mb Given that the heme electronic structure in Mb is affected by the nature of peripheral –R

PT

groups (see above), it follows that the thermodynamic and kinetic properties associated with modifying these substituents should also vary. For example, the basicity of the pyrrole nitrogens

RI

in the free porphyrin can be measured by the pK3 value, which is the equilibrium constant for the

SC

third protonation of porphyrin pyrrole nitrogen. This constant is dependent on the ability of the side chain groups of the porphyrin to donate or withdraw electron density. We correlate the pK3

NU

value of the free porphyrin to the basicity of the iron porphyrins studied in this work. In the series of hemes incorporated in the Mbs we investigated, the pK3 increases in the following

MA

order: diacetyldeuteroprotoporphyrin IX (3.3) < protoporphyrin IX (4.8) < deuteroporphyrin IX (5.5) < mesoporphyrin IX (5.8) [75]. The trend follows from the fact that the heme ethyl group present in the latter is the most electron releasing. More applicable to our studies is the pKa value

D

since it denotes a property of the heme enzyme instead of just the heme in solution. In particular,

TE

the pKa refers to the “acid-alkaline” transition (e.g. deprotonation/protonation of the water molecule bound to the heme at various pH) of the ferric form of the Mbs studied [34,76]. In hh

AC CE P

Mb, the pKa increases as follows: diacetyldeuteroMb (8.4; value obtained in this work) < wt Mb (8.7) < deuteroMb (8.8) < mesoMb (9.2) [76]. In this case, the basicity of the pyrrole nitrogens in the porphyrin affects the charge on the metal center, which is also manifested in its redox potential. Within the series of Mbs, the reduction potential decreases in the following order: wt Mb (-140 mV) > deuteroMb (-162 mV) > mesoMb (-170 mV) (reference electrode is Ag/AgCl) [38]. As discussed below, the kinetics of the reaction between MbII and NO2- correlates with the natural charge on Fe. Lastly, we determined experimental nitrite binding constants (Fig. S5), Knitrite, for ferric Mb, which we use to model the ferrous state since binding constants for the latter are difficult to achieve. Here, Knitrite decreases in the following order: diacetyldeuteroMb (132 M-1) > wt Mb (120 M-1) > deuteroMb (100 M-1) > mesoMb (90 M-1). Although the differences in the binding constant among the Mbs are not that large, the values reinforce the significance of the heme peripheral groups in stabilizing nitrite coordination. Here, the more electron donating groups on the 2,4-heme positions as in the case of mesoMb decrease the charge on the Fe center, destabilizing the nitrite complex. The opposite is true of diacetyldeuteroMb,

17

ACCEPTED MANUSCRIPT which has withdrawing substituents. A summary of the properties enumerated for the reconstituted Mb systems is presented in Table 3. As previously noted, the natural charge on Fe decreases in the following order as a result of

PT

the substituents present on the 2,4-heme positions: diacetyldeuteroMb > deuteroMb > wt Mb > mesoMb. Among the Mbs, diacetyldeuteroMb and mesoMb reduce NO2- the slowest and fastest,

RI

respectively, indicating that the rate of the reaction accelerates as the electron density on the Fe

SC

center increases (charge on metal center decreases) and the reduction potential decreases (Table 3, Figure 8). This trend is consistent with a previous study that demonstrates the rate of nitrite

NU

reduction in Mb being much faster than Hb, given that the redox potential of the former is lower (greater electron density on Fe) [7]. As seen in reaction (1), the rate limiting step involves the

MA

formation of MbIII as one of the products, which implies that the Mbs that are more easily oxidizable (lower reduction potential) form this product faster. Considering that mesoMb has the lowest reduction potential among the series of holoenzymes studied [38], it is expected to

D

generate the final products faster, which is consistent with our results. In the case of deuteroMb,

TE

since it rotates 180 about the - meso axis [42] and has the fastest heme reorientation rate among the systems studied [43], it is possible that its NiR activity is not simply attributed to the

AC CE P

Fe electron density brought about by the modifications on the 2,4-heme positions. It may also be influenced by the occurrence of multiple orientations within the heme pocket. The NiR activity of deuteroMb is therefore a reflection of the average activity of each orientational isomer. As previously suggested [26,71], the NiR mechanism in globins may depend on reprotonation of distal His and the energy barrier associated with this process. This step is therefore affected by the charge on the nitrite oxygen atom bound to the iron center (the O-atom that is more accessible to the distal His). For the N-bound complexes, the average natural charge of the nitrite oxygen atoms is as follows: -0.455 (mesoMb) < -0.451 (deuteroMb) < -0.441 (wt Mb) < -0.439 (diacetyldeuteroMb). In the case of the O-bound complexes, the natural charge for the nitrite O-atom (that accepts the proton) shows the following trend: -0.448 (mesoMb) < -0.445 (deuteroMb) < -0.436 (wt Mb) < -0.429 (diacetyldeuteroMb). Compared to diacetyldeuteroMb, the more negative charge on the nitrite O-atom in mesoMb in both linkage isomers signifies a higher tendency to accept the proton from the distal His residue. As this is thought to be a key step during the NiR reaction, this implies that mesoMb would have a faster reaction. This trend is consistent with our kinetics results. 18

ACCEPTED MANUSCRIPT The identity and stoichiometric amounts of the end products generated during the Mb NiR reaction of the different holoenzymes studied in this work provide insight into its chemistry. As shown in equations (1) and (2) [7,13], 1:1 stoichiometric amounts of MbIII and MbII-NO are

PT

produced upon the reaction of wt MbII with NO2-. Based on the fits to the final spectrum obtained after the reaction between nitrite and the ferrous forms of the different holoenzymes, the product

RI

is comprised of 1:1 MbIII and MbII-NO (Figure 5), which is consistent with previous studies

SC

[7,13]. Due to the high binding constant of NO to MbII [69] the ratio of the products formed will remain 1:1, particularly within the time scale of our studies. Within the variations induced by the

NU

heme –R groups presented in this work (e.g. natural charge on Fe center and O-atom of nitrite bound to heme, FeII-NHis93 and FeII-NPyr bond lengths; see discussion above), the relative

MA

amounts of MbIII and MbII-NO generated do not deviate from the stoichiometric ratio shown in equations (1) and (2). This suggests that the reconstituted Mbs may follow a mechanistic pathway similar to wt during the generation of NO via the NiR process.

TE

D

4.3. Correlation between the heme structure and energetics of MbII-nitrite intermediate Nitrite binding to MbII is an essential step in the mechanism of the NiR reaction, hence, we

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studied the nitrite binding affinity of the Mb model systems having various heme structures. Notably, the stability of the complexes may result from various factors such as the presence of His64 and other nearby amino acids stabilizing the heme pocket, kinetics of heme orientational disorder within the active site pocket (e.g. as in the case of deuteroMb), electronic interaction between the –R groups and the porphyrin  macrocycle, or perturbation of the heme environment (e.g. FeII-NHis93 bond) brought about by the –R substituents, among others. As recently discovered, the ferric Mb-nitrite intermediate exists in equilibrium between the nitro and nitrito forms [31]. To our knowledge, this phenomenon has not yet been probed for the ferrous form of the complex, however, it would most likely behave in a manner similar to the corresponding ferric complex where the intermediate can also interconvert between the N- and O-nitrite bound forms. Based on the relative binding energies of the N-bound complexes but with the exception of deuteroMb, diacetyldeuteroMbII-NO2- is the most stable among the series, while mesoMbII-NO2- is the least stable. In general, this comparison also holds true for the different Obound nitrite isomers (Table 2). Among the O-bound linkage isomers studied, the ΔEbinding for conformation 1 is ~2 kcal/mole more stable than the rest (Table 2), which has also been observed 19

ACCEPTED MANUSCRIPT in previous reports for the ferric nitrite complex [39]. Although our calculations suggest that Nbound complexes are favored, which on average is more stable than the nitrito complex by ~5.0 kcal/mole, the possible presence of both conformations in solution cannot be discounted during

PT

the NiR reaction. This is supported by a recent study in which it was observed that both the Oand N-bound ferric Mb-nitrite complexes exist in solution as a result of the fast binding

RI

equilibrium between the two conformers, with the nitro form being more stable by several

SC

kilocalories/mole [31]. Crystal structures of both N- and O-bound Mb ferrous and ferric nitrite linkage isomers [28,29,32] further reinforce this idea. In addition, QM/MM studies of nitrite

NU

binding to ferric Hb indicate a small energy difference between the nitro and nitrito conformers, suggesting the possible presence of both linkage isomers in solution [26]. With the exception of

MA

deuteroMb (deuteroMb is unique in that its NiR function may be influenced by the presence of multiple heme conformation), a correlation is observed between the stability of the nitrite complex and the bimolecular rate constant. Here, the more stable the intermediate, the slower the

D

NiR reaction. Further, results show that the nitrite binding equilibrium constant of ferric Mb

TE

decreases as the nitrite binding energy (of ferrous Mb) increases (Table S3). Assuming the trend of Knitrite in ferric Mb is applicable to the ferrous form, it can be inferred that the NiR rates are

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influenced by the heme redox potential more than the binding affinity of nitrite. The differences in the nitrite binding energies of the various Mbs can be attributed to the electronic effects of the substituents at the 2,4-heme positions. The more electron-withdrawing substituents tend to increase the natural charge of iron, resulting in a more stable complex upon binding of the negatively charged nitrite ligand. This is demonstrated in the series of Mbs studied here, where diacetyldeuteroMb (more electron-withdrawing substituent) generates a more stable complex than that of mesoMb (less electron withdrawing –R group). Aside from the natural charge on Fe, the heme geometric structure of the nitrite-bound complex provides insight into the differences in the nitrite binding ability of the various Mbs. A closer look into the FeII-NHis93 and FeII-NO2-/FeII-ONO- bond lengths reveal an inverse relationship (Table 1). Taking the case of diacetyldeuteroMb and mesoMb, whose electronic structures are the most disparate among the Mbs investigated, it is noted that the FeII-NHis93 bond length is longer in the former than the latter when complexed with nitrite (e.g. 2.031 Å vs 2.016 Å in nitrito conf1); however, the FeII-ONObond length shortens (e.g. 1.974 Å vs 1.984 Å in nitrito conf1). This phenomenon is consistent among the Mb samples and nitrite/nitrito conformations studied, and parallel those observed for 20

ACCEPTED MANUSCRIPT six-coordinate ferrous nitrosyl compounds with N-donor ligands that exhibit weakening of the FeII-NO and N-O bond as the FeII-N(donor) bond strengthens [77,78]. Our work demonstrates a direct correlation between the FeII-nitrite bond length and the stability of the complex: the

PT

stronger the FeII-NO2-/FeII-ONO- bond, the more stable the complex. On average, diacetyldeuteroMb shows the shortest (strongest) FeII-NO2-/FeII-ONO- bond and is calculated to

RI

have the lowest nitrite binding energy (most stable complex).

SC

Beyond the heme electronic structure, the presence of amino acid residues on the distal side in close proximity to the binding ligand provides further stability. While nearby residues Ile107

NU

and Phe43 are close enough to non-covalently interact with small ligands on the iron center, His64 is crucial as it directly interacts with small ligands and provides H-bonding interaction.

MA

Native Mb binds nitrite O-bound, but when His64 is mutated to Val, devoid of H-bonding interaction, nitrite flips to the N-bound form; reincorporation of a H-bonding residue in the form of a double mutant, H64V/V67R, isomerizes nitrite back to O-bound [32]. This distal residue

D

therefore modulates the conformation, and hence the energetics of nitrite binding to the iron

TE

center as further supported by quantum calculations [30,39]. Interestingly, neuroglobin (Ngb), a six-coordinate globin that catalyzes the reduction of NO2- to NO, is shown to also be affected by

AC CE P

mutations induced on the distal heme pocket, but for reasons different from that of Mb. While nitrite reduction in Mb is facilitated through H-bonding interaction, reaction rates in Ngb are related to the distal pocket volume and heme accessibility, which correlate with the absence of distal H64. This is supported by markedly much faster rates observed in H64A Ngb variant (1120 M-1 s-1) compared to wt Ngb (0.52 M-1 s-1) [79], where the distal pocket volume of the former mutant is larger than the latter. Mutating H64 generates a penta-coordinate heme, allowing nitrite to readily bind to the iron center as the coordination site opens up. The phenomenon observed in Ngb is different from Mb, where H64 Mb mutants show slower rates than wt Mb [32]. The absence for the need of H64 in Ngb suggests that the mechanism of nitrite reduction in Ngb does not rely on the delivery of a proton from the H64 sidechain unlike that of penta-coordinated globins [26], and uses an alternate mechanism instead. Since globins require distal H64 during the NiR reaction, it may be implied that protonation of this residue is the limiting step in the reaction. Computational data support this [26], but have yet to be experimentally verified.

21

ACCEPTED MANUSCRIPT 4.4. A broader chemical insight into globin NiR activity During the NiR reaction in globins, nitrite is one electron reduced to NO followed by a one or

PT

two proton transfer process depending on the identity of the ligand (e.g. nitrite versus nitrous, Scheme 1). While the most energetic barriers are proposed to stem from either the reprotonation

RI

of distal His or the dissociation of NO from ferric heme [26], we focus on the structural and electronic properties of the elusive MbII-nitrite intermediate and its influence on the NiR activity.

SC

At the geometric structural level, the NiR activity is not affected by variations in the FeII-NPyr and FeII-NHis93 bond lengths of the MbII-nitrite intermediate. Typically, the strength of the FeII-

NU

NHis93 bond controls the ligand reacting trans to this proximal residue, but this is not the case during the reduction of nitrite by Mb despite the observed inverse correlation between the Fe II-

MA

nitrite and FeII-NHis93 bond lengths as would be the expected effect of N-donor ligands. A closer look into the natural charges of key atoms indicates a different story. For example, the rate of

D

nitrite reduction speeds up as the natural charge on the Fe center decreases (reduction potential

TE

decreases), indicating that the rate is dependent on the ease by which the heme is oxidized. While this relationship is consistently demonstrated in a similarly penta-coordinated Hb [7,17], hexa-

AC CE P

coordinated Ngb shows a poor correlation of redox potential (which is affected by Fe center charge) with NiR activity (see discussion above, [79]). Beyond the charge of the Fe center, the charge on the oxygen atom of nitrite (that accepts a proton) in the MbII-nitrite intermediate affects the reaction rate. Since the reaction mechanism involves the protonation of nitrite and the reprotonation of distal His as potentially the process with the greatest energy barrier [26], the correlation of the natural charge on the oxygen atom with the NiR activity as shown in our work supports this idea. The relationship between the aforementioned parameters to the NiR reaction rates apply both to the nitro and nitrito-bound intermediate. As shown in Scheme 1, the conformation of this complex and the type of ligand bound to the Fe center dictates the product formed, and therefore the geometry of the intermediate is a key matter of discussion. For example, the nitro bound intermediate will form MbII-NO, whereas the nitrito bound complex yields MbIII-OH [26]. Our calculations indicate a low energy difference between the linkage isomers, alluding to the idea that a more complicated process involving the MbII-intermediate linkage isomers is plausible.

22

ACCEPTED MANUSCRIPT 5. Conclusions In this work, we explore the effect of systematically modifying the 2,4-heme position on the NiR activity of globins using reconstituted Mbs as a model system. Results demonstrate a

PT

correlation between the natural charge on the Fe center and O-atom of nitrite bound to heme that is free to accept protons, the nitrite binding energies, and the bimolecular rate constant of the

RI

reaction between MbII and NO2- for the series of Mb samples investigated. In particular, NiR

SC

activity of the Mbs investigated in this work increase in the following order: diacetyldeuteroMb < wt Mb < deuteroMb < mesoMb. The stoichiometry of the products of the NiR reaction, Mb III

NU

and MbII-NO, does not vary among the Mb samples. This demonstrates that the geometric and electronic structural modifications induced by the various hemes studied in our work have minimal effect on the mechanistic pathway of the NiR process in five-coordinate globins. Our

MA

calculations on the elusive FeII-nitrite species imply that the N- and O-bound nitrite linkage isomers may both exist during the NiR reaction. Further, the nitrite binding energies of the

D

different O-bound conformers are close enough such that one might envision the occurrence of

TE

these isomers simultaneous flipping within the heme pocket, alluding to a more complicated

AC CE P

mechanistic reaction than initially conceived and hence requires further investigation. ACKNOWLEDGEMENTS

MGIG would like to acknowledge Sigma Delta Epsilon-Graduate Women in Science, and the Spectroscopy Society of Pittsburgh for financial support. RSF and AS thank Penn State Behrend for fellowship support. JFG acknowledges support from the Welch Foundation (BD-0046), Laboratory for Molecular Simulation at Texas A&M University for providing the Gaussian 09 software, and Texas A&M Supercomputing Facility (http://sc.tamu.edu) for computer time.

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P. Kleinbongard, et al., Free Rad. Biol. Med. 35 (2003) 790-796.

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[79]

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Scheme 1. Proposed mechanisms (1-3) for the reduction of NO2- to NO in five-coordinate

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globins [26]. In our work, we determine only the nitrite binding energies of the N- and O-bound ferrous complexes as a function of porphyrin substitution. Our experimental conditions (pH 7.4)

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will predominantly contain the nitrite ligand.

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Figure 1. Structures of A) diacetyldeuteroheme, B) protoheme (present in wt Mb), C) deuteroheme, and D) mesoheme. The electron releasing capacity of the —R groups in positions

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l-and 4- increase on going from left to right. The unnatural hemes were reconstituted into

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apoMb.

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Figure 2. Different orientations of the MbII-nitrite complex simulated in our studies: A) Nbound, B) O-bound conformation 1, C) O-bound conformation 2, D) O-bound conformation 3.

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The representative optimized structures shown are based on cd1 bacterial nitrite reductase (Nbound) [27] and Hb (O-bound conformations) [47]. The red, blue, purple, and gray spheres represent oxygen, nitrogen, iron, and carbon atoms, respectively.

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200

II

150

Mb -NO

12

80

4

-1

500

600

700

120

0

SC

80

600

400 500 600 Wavelength/nm

500

700

700

40 0

600

700

III

Mb II Mb III Mb -NO2

160 120

500

0

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II

Mb -NO

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5

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Mb II Mb III Mb -NO2

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III

10 8 6 4 2 0

15

50

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/mM cm

0

II

Mb -NO

20

100

8

40

B

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A

III

Mb II Mb III Mb -NO2

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III

Mb II Mb III Mb -NO2

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Mb -NO 12 8 4 0

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400 500 600 Wavelength/nm

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Figure 3. UV-visible spectra of MbIII (black, solid line), MbII (green, short dotted line), MbIIINO2- (red, dashed line), and MbII-NO (blue, dash dotted line) forms of A) diacetyldeuteroMb, B) wt Mb, C) deuteroMb, and D) mesoMb in 100 mM phosphate buffer, pH 7.4.

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diacetyldeuteroMb

II

0.6

0.4

0.4

0.4

0.2 0.0 400

0.2

425

450

475

0.0

wt Mb

400

425

450

0.8

500

0.02 0.00

412

500

600

II

Mb II Mb + NO2 product

434

554

0.08

503

582

0.04

0.4

700

549

0.00 500

600

700

0.0

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0.0

deuteroMb

0.8 0.6 0.4 0.2 0.0 360 380 400 420 440

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0.4 0.0

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mesoMb

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Absorbance

0.4

0.8

0.04 0.00

552

0.04

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0.8 0.6 0.4 0.2 0.0 375

0.06

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561

430

0.2 0.0

Mb II Mb + NO2 product

448

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0.6

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0.0 300

400

500

0.4

0.04

567 621

0.00

500

600

700

II

Mb II Mb + NO2 product

421 0.04

0.4 380

543 531

0.6

0.2

0.2

0.08

396

0.4

0.0 360

397 421

0.0

0.6

0.4

0.8

II

Mb II Mb + NO2 product

400

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Wavelength/nm

420

440

700

532

0.2 0.0 300

544

490

569 624

0.00 500

400

500

600

600

700

700

Wavelength/nm

Figure 4. Left panel: Time-evolution spectral changes showing the conversion of MbII (5-8 M) to the products, MbIII and MbII-NO, upon addition of stoichiometric amounts of nitrite (diacetyldeuteroMb, 224 min; wt Mb, 209 min; deuteroMb, 75 min; mesoMb, 75 min). The insets focus on the isosbestic changes in the Soret region. Sample kinetic traces are shown in Figure S2. Right panel: Absorption spectra of Mb II (black, dashed line) and the products (red, solid line) generated after the NiR reaction. Absorption spectra were taken in 100 mM phosphate buffer, pH 7.4. 32

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0.6 0.08

0.4

B 1.2 0.12

0.8

0.08

0.04

0.02

0.2

500

600

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400 500 600 700 Wavelength/nm

0.0

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Experimental spectrum Sum of reference spectra III 50% + 2% Mb II 50% + 2% Mb -NO

0.04 0.02 0.00

500

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400 500 600 700 Wavelength/nm

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700

Experimental spectrum Sum of reference spectra III 50% + 2% Mb II 50% + 2% Mb -NO

0.8 C 0.6

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Absorbance

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Experimental spectrum Sum of reference spectra III 49% + 2% Mb II 49% + 2% Mb -NO II 2% + 2% Mb

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Experimental spectrum Sum of reference spectra III 49% + 2% Mb II 47% + 3% Mb -NO II 4% + 4% Mb

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Figure 5. Absorption spectra of A) diacetyldeuteroMb, B) wt Mb, C) deuteroMb, and D) mesoMb in phosphate buffer, pH 7.4. The reference spectra of MbIII (blue, dash dotted line), MbII-NO (green, short dotted line), and MbII (purple, dotted line); and the experimental spectrum (black solid line) and the sum of the reference spectra (red, dashed line) are shown. Results show the contribution of each species to the final experimental spectrum. All data were collected in 100 mM phosphate buffer, pH 7.4.

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diacetyldeuteroMb

wt Mb 3

2

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1

k = 1.2 + 0.1 M-1 s-1 0 k = 2.2 + 0.2 M-1 s-1 0 2 4 6 8 10 12 14 16 18 0 2 4 6 8 10 12 14 16 18 8

deuteroMb

6 5

mesoMb

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kobs/s-1 x 10-3

1

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3

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2

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1 k = 4.9 + 0.3 M-1 s-1 k = 7.4 + 0.4 M-1 s-1 0 0 0 2 4 6 8 10 12 14 16 18 0 1 2 3 4 5 6 7 8

[NO2-]/M x 10-4

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Figure 6. Observed reaction rates for the different MbII (4-6 M) samples as a function of nitrite

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concentration at 20 C. The linear fit of each plot generates the bimolecular rate constant for the particular Mb sample. The solutions were prepared in 100 mM phosphate buffer, pH 7.4.

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Figure 7. The relevant σ Fe-NO2 natural bond orbitals of the six-coordinate Fe-NO2- complexes of A) diacetyldeuteroMb, B) wt Mb, C) deuteroMb, and D) mesoMb.

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Figure 8. Correlation between the natural charge on the Fe center (top), and the reduction potential (bottom) on the bimolecular rate constant of the reaction between MbII and NO2- for the Mb samples investigated (square symbol). The corresponding heme structure for each holoenzyme is presented beside the symbol.

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N-bound O-bound conf1 O-bound conf2 1.942 1.974 1.991 1.947 1.977 1.996 1.939 1.980 2.000 1.952 1.984 2.007

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S=2 2.092 2.089 2.092 2.079

Structure

diacetyldeuteroMb wt Mb deuteroMb mesoMb

S=0 1.915 1.907 1.913 1.902

-

with NO2 N-bound

S=2 2.100 2.095 2.107 2.100

2.137 2.118 2.146 2.137

2.031 2.039 2.030 2.016

2.050 2.039 2.046 2.030

with NO2O-bound conf3 2.024 2.008 2.018 2.002

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The abbreviation “conf” pertains to “conformation”.

O-bound conf3 1.959 1.964 1.964 1.971

FeII-NHis93 distances with NO2with NO2O-bound conf1 O-bound conf2

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without NO2

-

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S=0 2.032 2.034 2.036 2.046

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Table 1. Geometrical parameters of optimized models MbII and MbII-nitrite complexes. Structure Distances (Å) Average FeII-NO2- or FeII-ONO- distances II Fe -Npyr (without NO2-)

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Table 2. Relative nitrite binding energies of the different conformations of MbII-nitrite model complexes. Structure Binding Energies (kcal/mole) N-bound O-bound conf1 O-bound conf2 O-bound conf3 E1 1 E22 E11 E22 E11 E22 E11 E22 diacetyldeuteroMb 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 wt Mb 1.76 1.12 5.24 3.87 5.22 4.54 4.29 3.47 deuteroMb 8.67 8.33 8.17 7.96 9.30 8.13 7.96 7.64 mesoMb 6.75 2.40 7.16 5.23 8.10 5.93 7.16 4.92 1

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E1= [EMb(II)-NO2 at S=0 – (EMb(II) at S=0 + ENO2)], the energies of the unbound state was calculated with spin state S=0. E2= [EMb(II)-NO2 at S=0 – (EMb(II) at S=2 + ENO2)], the energies of the unbound state was calculated with spin state S=2. All the reported energies were calculated relative to the lowest nitrite binding energy for each conformation among the Mb models.

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Table 3. Thermodynamic and kinetic parameters for the reconstituted Mb samples in 100 mM phosphate buffer, pH 7.4. Parameters Mb samples diacetyldeuteroMb wt Mb deuteroMb mesoMb refs. pK31 3.3 4.8 5.5 5.8 [75] pKa2 8.43 8.7 8.8 9.2 [76] -1 Knitrite (M ) 132 120 100 90 this work4 Reduction N/A -140 -162 -170 [38]5 potential (mV) k (M-1 s-1) 1.2 + 0.1 2.2 + 0.2 4.9 + 0.3 7.4 + 0.4 this work6 Enitrite (kcal/mol) 0.00 1.12 8.33 2.40 this work7 pK or basicity of the pyrrole nitrogens in the free porphyrin. 2“acid-alkaline” transition (protonation/deprotonation of the heme-bound water molecule) in metmyoglobin. 3Determined in this work. 4The nitrite binding equilibrium constant pertains to nitrite binding to ferric Mb. Also see Fig. S5. The value for ferric wt Mb was determined from ref. [33]. 5The reference electrode is Ag/AgCl. 6Rates were determined at 20C. 7Relative nitrite binding energy of the nitro isomer.

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Graphical Abstract

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The effect of the 2,4-heme substituents on the nitrite reductase (NiR) activity of myoglobin (Mb) was investigated by reconstituting unnatural hemes into apoMb. Holoenzymes with hemes containing more electron releasing groups (lower Fe charge) show higher NiR activity over those that have more electron withdrawing substituents (higher Fe charge).

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Highlights  The nitrite reductase activity of Mb is affected by varying the 2,4-heme substituents.  DFT calculations show the nitro MbII-NO2- complex is more stable than the nitrito form.  The nitrite binding energies to ferrous Mb are affected by the 2,4-heme substituents.

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