The distal pocket histidine residue in horse heart myoglobin directs the O-binding mode of nitrite to the heme iron

Abstract

It is now well-established that mammalian heme proteins are reactive with various nitrogen oxide species and that these reactions may play significant roles in mammalian physiology. For example, the ferrous heme protein myoglobin (Mb) has been shown to reduce nitrite (NO(2)(-)) to nitric oxide (NO) under hypoxic conditions. We demonstrate here that the distal pocket histidine residue (His64) of horse heart metMb(III) (i.e., ferric Mb(III)) has marked effects on the mode of nitrite ion coordination to the iron center. X-ray crystal structures were determined for the mutant proteins metMb(III) H64V (2.0 A resolution) and its nitrite ion adduct metMb(III) H64V-nitrite (1.95 A resolution), and metMb(III) H64V/V67R (1.9 A resolution) and its nitrite ion adduct metMb(III) H64V/V67R-nitrite (2.0 A resolution). These are compared to the known structures of wild-type (wt) hh metMb(III) and its nitrite ion adduct hh metMb(III)-nitrite, which binds NO(2)(-) via an O-atom in a trans-FeONO configuration. Unlike wt metMb(III), no axial H(2)O is evident in either of the metMb(III) mutant structures. In the ferric H64V-nitrite structure, replacement of the distal His residue with Val alters the binding mode of nitrite from the nitrito (O-binding) form in the wild-type protein to a weakly bound nitro (N-binding) form. Reintroducing a H-bonding residue in the H64V/V67R double mutant restores the O-binding mode of nitrite. We have also examined the effects of these mutations on reactivities of the metMb(III)s with cysteine as a reducing agent and of the (ferrous) Mb(II)s with nitrite ion under anaerobic conditions. The Mb(II)s were generated by reduction of the Mb(III) precursors in a second-order reaction with cysteine, the rate constants for this step following the order H64V/V67R > H64V >> wt. The rate constants for the oxidation of the Mb(II)s by nitrite (giving NO as the other product) follow the order wt > H64V/V67R >> H64V and suggest a significant role of the distal pocket H-bonding residue in nitrite reduction.

Figure 1

Crystallographically determined binding modes of nitrite to heme proteins and copper enzymes.

Figure 2

Sketches of the active sites of wild-type (wt) and mutant Mbs.

Figure 3

The 2F_o−F_c electron density maps (left; contoured at 1σ) and F_o−F_c electron density map (right; contoured at 3σ) and final models of the heme environments in the crystal structures of the ferric hh Mb H64V single mutant. The 1.95 Å resolution crystal structure of the nitrite adduct of this H64V mutant is shown in A and B (PDB access code 3HEP). The 2.0 Å resolution crystal structure of the ferric H64V mutant without nitrite is shown in C and D (PDB access code 3HC9).

Figure 4

The 2F_o−F_c electron density maps (left; contoured at 1σ) and F_o−F_c electron density map (right; contoured at 3σ) and final models of the heme environments in the crystal structures of the ferric hh Mb H64V/V67R double mutant. The 2.0 Å resolution crystal structure of the nitrite adduct of this H64V/V67R mutant is shown in A and B (PDB access code 3HEO). The 1.90 Å resolution crystal structure of the ferric H64V/V67R double mutant without nitrite is shown in C and D (PDB access code 3HEN).

Figure 5

The trans-FeONO and cis-FeONO conformations.

Figure 6

Superposition of the final models of the active sites of the ferric hh Mb H64V/V67R (light gray) and that of the ferric hh Mb H64V/V67R-nitrite complex (dark gray) structures. The nitrite ligand is not shown.

Figure 7

UV-vis spectroscopic monitoring of the reactions of the metMbs with excess cysteine (1 mM) under anaerobic conditions in 0.1 M phosphate buffer at pH 7.41 at 25 °C. A: [wt] = 3.7 µM. B: [H64V] = 3.7 µM. C: [H64V/V67R] = 3.5 µM.

Figure 8

The reaction between cysteine reduced wt Mb^II (2.5 µM) and nitrite (1 mM) was monitored by UV/vis at 25 °C in 0.1 M phosphate buffer at pH 7.41. The loss in absorbance for Fe^II at 433 nm was used to calculate the initial rates. After Fe^II oxidation in excess cysteine the reaction proceeds to form Fe^II(NO) on the same timescale as the rate limiting cysteine reduction.

Scheme 1