Intermediates Involved in the Two Electron Reduction of NO to N₂O by a Functional Synthetic Model of Heme Containing Bacterial NO Reductase

Nitric Oxide (NO) is a freely diffusing hormone that is an integral component of the physiology of all advanced life forms. It is involved in regulation of metabolism, blood flow, immune responses and apoptosis.¹ NO is also an intermediate in bacterial denitrification (i.e. reduction of NO₃⁻ to N₂) and is further reduced to N₂O by the enzyme nitric oxide reductase (NOR).² Bacterial NOR’s are membrane bound enzymes that are members of the heme-copper oxidase super-family. NORs are proposed to have a bimetallic heme/non-heme catalytic active site with two electron transfer sites quite like the cytochrome c oxidases (CcO).³ While heme a₃ in NORs is proposed to be similar to that in CcO, the distal metal in NORs is Fe (referred to as Fe_B) instead of Cu_B in CcO. Also the post-translationally crosslinked Tyr244-His ligand in CcO is absent in NORs; rather a few glutamate residues are proposed to play an important role in NOR’s reactivity.³

In the absence of structural data on these enzymes, much of the available insight has been acquired via spectroscopic probes and biochemical studies. Not surprisingly the mechanism of action of NORs is a matter of debate. Spectroscopic evidence suggests that the reduction of NO to N₂O involves a bis-nitrosyl intermediate where each of the reduced (ferrous) irons is bound to a molecule of NO.⁴ Then N-N bond formation and further rearrangement lead to the formation of N₂O and a bis-ferric resting active site.⁴ This mechanism has not been supported by synthetic model complexes of NOR, such as the one reported by Karlin and co-workers, as such a bis-nitrosyl complex was found to be either inactive or it did not stoichimetrically form N2O.^5a,5b,6 An alternate cismechanism has been proposed where two NO molecules bind the distal metal and then undergo reductive cleavage forming N₂O.⁷

Recently a synthetic functional model of NOR has been reported by Collman et.al. where a bis-ferrous heme non-heme model (Fe^II_H/Fe_B^II) reduces two molecules of NO to generate N₂O (Scheme 1).⁸ In the present study we report two nitrosyl intermediates formed along the reaction of this functional NOR model and characterize them using electron paramagnetic resonance (EPR) and resonance Raman (rR) spectroscopy.

Scheme 1.

A synthetic functional model of NOR. It includes a tris-imidazole distal pocket that binds Fe_B and a porphyrin macrocycle with a covalently attached proximal imidazole that binds Fe_H.

The low-spin S=1/2 Fe_H^II-NO has a characteristic EPR signal around 3200 Gauss (g= 2.07, 2.02, 1.96; Fe_H^II-NO/Zn_B in Fig.1 black)⁹ which is very different from the EPR signal of the S=3/2 Fe_B-NO around 1700 Gauss (g=3.92; Fe_H^II/Fe_B-NO in Fig.1 pink).¹⁰ The rR spectrum of the Fe_H^II-NO/Zn_B show that the Fe_H^II-NO stretch at 581 cm⁻¹ shifts to 545 cm⁻¹ upon ¹⁵NO (Fig. 2) substitution. The 239 cm⁻¹ mode (Fig. 2) is possibly a Fe_H^II-N_Imz vibration as it is lowered from 242 cm⁻¹ in the Fe_H^II complex upon NO binding (Fig. S1) forming a six coordinate complex.⁹ The N-O vibration is found (FTIR, Fig. S2) to be at 1635 cm⁻¹ and shifts to 1590 cm⁻¹ upon ¹⁵NO substitution. Furthermore the porphyrin υ₄ band, which is very sensitive to the charge and spin state of the bound heme Fe,¹¹ is observed at 1357 cm⁻¹ and 1365 cm⁻¹ (Fig. 3A) for Fe_H^II and Fe_H^II-NO, repectively. These characteristic EPR and rR features are used to identify the reaction intermediates.¹²

Figure 1.

EPR of the intermediates and final product in the reaction of NO with the bis-ferrous model. In the legend H=heme and B=distal pocket. All samples are 1mM in CH₂Cl₂, T=4K and microwave power = 10mW.

Figure 2.

rR data for the heme Fe-NO species ¹⁴NO (blue) and ¹⁵NO (red)

Figure 3.

rR data of the starting bis-ferrous complex, intermediates and end product A) high-energy region (includes heme-Fe^II-NO data for reference) and B) low-energy region.

Figure 1 shows the EPR spectrum obtained by adding NO to the bis-ferrous complex in dichloromethane solution at different temperatures. At −80°C (intermediate A) the spectrum has a signal at 1700 Gauss (g=3.92, Fig.1, red) which is identical to the Zn_H/Fe_B-NO complex both in energy and intensity (Fig.1, pink), implying that this is a mono-nitrosyl Fe_B-NO species. The rR data of intermediate A in the high-energy region (Fig. 3 A, red) shows υ₄ band at 1356.5 cm⁻¹ characteristic of a Fe_H^II (1357 cm⁻¹) indicating that there is no reaction at the Fe_H^II in this intermediate. No vibrational data on the Fe_B-NO could be obtained due to its weaker enhancement relative to heme vibrations. When the reaction is warmed to −40°C (intermediate B), no signal is observed in the EPR spectrum. The same EPR data can also be obtained when the reaction is rapidly frozen at room temperature. Intermediate B shows a υ₄ band at 1366.1 cm⁻¹(Fig. 3A, blue), ~8cm⁻¹ higher than intermediate A showing that NO is now bound to Fe_H. There’s also a band at 1620 cm⁻¹ that shifts to 1579 cm⁻¹ when ¹⁵NO was used (Fig. S3). In the low energy region a band is observed (Fig. 3 B) at 585 cm⁻¹ (blue) that shifts to 559 cm⁻¹ with ¹⁵NO (red) while another band is observed at 460 cm⁻¹ which shifts to 432 cm⁻¹ with ¹⁵NO substitution. The 585 cm⁻¹ band is assigned as Fe_H^II-NO stretching vibration based on data obtained with the Fe_H^II-NO complex (Fig. 2). The band at 432 cm⁻¹ is consistent with a Fe-N-O bending mode (an Fe_B-N vibration can not be ruled out). These data indicate the presence of a Fe_H^II-NO center in intermediate B.

Warming the reaction mixture up to room temperature results in a heme-Fe^III-NO/Fe_H^III-OH product (EPR in Fig.1, light green).⁸ Intermediate A is a Fe_H^II/Fe_B-NO complex as indicated by the υ₄ band of the Fe_H^II and the S=3/2 EPR signal of the Fe_B-NO. Intermediate B has a Fe_H^II-NO center in it as indicated by the Fe-N vibration (Fig. 3B) and N-O vibration (Fig. S3). Note that both of these vibrations are different from that of an isolated Fe_H^II-NO/Zn^II_B complex. The Fe-N vibration is higher (589 cm⁻¹ in B, 581 cm⁻¹ in Fe_H^IINO) and the N-O vibration is lower (1620 cm⁻¹ in B 1635 cm⁻¹ in Fe_H^IINO). Thus intermediate B could either be 1) a mononitrosyl derivative where the NO dissociates from the Fe_B and binds to heme-Fe at higher temperatures or 2) a bis-nitrosyl derivative.¹³ The reaction of the same bis-ferrous complex in N,N-dimethylformamide (DMF) results in a bis-nitrosyl species where both S=3/2 (Fe_B-NO) and S=1/2 (Fe_H^II-NO) EPR signals (similar to those reported by Shiro et.al.)⁴ and a heme-Fe-N vibration at 572 cm⁻¹ (unperturbed relative to that for the Fe_H-NO/Zn_B complex, Fig. 2) are observed (Fig. S4, S5). However, this reaction does not yield N₂O. Also the Zn_H^II/Fe_B-NO species shows no NO dissociation at room temperature. Thus formation of an EPR active but functionally inactive bis-nitrosyl in DMF, the stability of Fe_B-NO, perturbation of the heme-nitrosyl vibrational features, and the lack of an EPR signal indicate that the intermediate B is a bis-nitrosyl complex where the two nitrosyls are close enough to either spin-dipolar couple or are directly interact. A very similar situation was reported for the heme non-heme bis carbonyl complex for the active site of NOR.¹⁴

Scheme 2.

Proposed Mechanism of N₂O Formation from NO. These results support a trans-mechanism of N₂O formation by this synthetic functional model of NOR (sheme 2). The first NO binds to Fe_B forming a high-spin Fe_B-NO species (A), followed by another NO binding to heme Fe^II resulting in the formation of a Fe_H^II-NO/Fe_B-NO species (B) which then forms N₂O and a bis ferric product. This study provides first direct evidence for a trans bis nitrosyl intermediate as a viable pathway for the two electron reduction of NO to N₂O by the bis ferrous active site of NOR.