Effect of Outer-Sphere Side Chain Substitutions on the Fate of the trans Iron-Nitrosyl Dimer in Heme/Nonheme Engineered Myoglobins (Fe(B)Mbs): Insights into the Mechanism of Denitrifying NO Reductases

Abstract

Denitrifying NO reductases are transmembrane protein complexes that utilize a heme/nonheme diiron center at their active sites to reduce two NO molecules to the innocuous gas N2O. Fe(B)Mb proteins, with their nonheme iron sites engineered into the heme distal pocket of sperm whale myoglobin, are attractive models for studying the molecular details of the NO reduction reaction. Spectroscopic and structural studies of Fe(B)Mb constructs have confirmed that they reproduce the metal coordination spheres observed at the active site of the cytochrome c-dependent NO reductase from Pseudomonas aeruginosa. Exposure of Fe(B)Mb to excess NO, as examined by analytical and spectroscopic techniques, results primarily in the formation of a five-coordinate heme-nitrosyl complex without N2O production. However, substitution of the outer-sphere residue Ile107 with a glutamic acid (i.e., I107E) decreases the formation rate of the five-coordinate heme-nitrosyl complex and allows for the substoichiometric production of N2O. Here, we aim to better characterize the formation of the five-coordinate heme-nitrosyl complex and to explain why the level of N2O production increases with the I107E substitution. We follow the formation of the five-coordinate heme-nitrosyl inhibitory complex through the sequential exposure of Fe(B)Mb to different NO isotopomers using rapid-freeze-quench resonance Raman spectroscopy. The data show that the complex is formed by the displacement of the proximal histidine by a new NO molecule after the weakening of the Fe(II)-His bond in the intermediate six-coordinate low-spin (6cLS) heme-nitrosyl complex. These results lead us to explore diatomic migration within the scaffold of myoglobin and whether substitutions at residue 107 can be sufficient to control access to the proximal heme cavities. Results on a new Fe(B)Mb construct with an I107F substitution (Fe(B)Mb3) show an increased rate for the formation of the five-coordinate low-spin heme-nitrosyl complex without N2O production. Taken together, our results suggest that production of N2O from the [6cLS heme {FeNO}(7)/{Fe(B)NO}(7)] trans iron-nitrosyl dimer intermediate requires a proton transfer event facilitated by an outer-sphere residue such as E107 in Fe(B)Mb2 and E280 in P. aeruginosa cNOR.

Figure 1

Active site structures of Fe_BMb1 (PDB entry 3K9Z) and Fe_BMb2 (PDB entry 3M39).(14, 15)

Figure 2

RR spectra of RFQ samples of the reaction of Fe_BMb1(¹⁴NO) with a 3-fold ¹⁵NO excess (excitation wavelength 406 nm, sample temperature 110 K).

Figure 3

Room temperature UV-vis absorption spectra of reduced Fe_BMb3 after loading of the Fe_B site with Fe(II) (black) and of the resting complex formed after exposure to 100 μM NO (red).

Figure 4

RR spectra of the reaction end product of reduced Fe_BMb3 with 4-fold ¹⁴NO excess (black traces) and ¹⁵NO (red traces). Also shown are ¹⁴NO – ¹⁵NO differential signals for the nonheme ν(NO), heme ν(NO), and heme ν(FeNO) modes (blue traces) (excitation wavelength 406 nm, sample temperature 110 K).

Figure 5

RR spectra of RFQ samples of the reaction of reduced Fe_BMb3 with 3-fold ¹⁴NO excess (black traces) or ¹⁵NO (red traces) compared with those of resting reduced Fe_BMb3 (gray traces) (excitation wavelength 406 nm, sample temperature 110 K). Also shown are selected ¹⁴NO – ¹⁵NO differential signals for the nonheme ν(NO) and heme ν(FeNO) modes (blue traces).

Figure 6

Stopped-flow UV-vis absorption spectra of the reaction of reduced Fe_BMb3 with 70 μM and 1 mM NO at 4 °C. Also shown are the dependence of observed rate constants on NO concentrations.

Figure 7

Room-temperature RR spectra of the iron(II)-loaded reduced protein Fe_BMb1 (black), Fe_BMb2 (red), and Fe_BMb3 (blue) obtained with a 442-nm laser excitation.

Scheme 1

Reaction of reduced Fe_BMbs with NO.