Solvent Accessibility in the Distal Heme Pocket of the Nitrosyl d1-Heme Complex of Pseudomonas Stutzeri cd1 Nitrite Reductase.

In nitrite reductase (cd1 NIR) the c-heme mediates electron transfer to the catalytic d1-heme where nitrite (NO2-) is reduced to nitric oxide (NO). An interesting feature of this enzyme is the relative lability of the reaction product NO bound to the d1-heme. Marked differences in the c- to d1-heme electron-transfer rates were reported for cd1 NIRs from different sources, e.g. Pseudomonas stutzeri (P. stutzeri) and Pseudomonas aeruginosa (P. aeruginosa). While the three dimensional structure of the P. aeruginosa enzyme has been determined, that of the P. stutzeri enzyme is still unknown. The difference in electron transfer rates prompted us to compare the structural properties of the d1-heme pocket of P. stutzeri cd1 NIR with those of the P. aeruginosa wild type enzyme (WT) and its Y10F using their nitrosyl d1-heme complexes. We applied high field pulse Electron Paramagnetic Resonance (EPR) techniques that detect nuclear spins in the close environment of the spin bearing Fe(II)-NO entity. We observed no significant differences in the rhombic g-tensor and detected a proximal histidine ligand with 14N hyperfine and quadrupole interactions similar to those of P. aeruginosa WT and Y10F mutant complexes. We did observe significant differences in the H-bond network involving the NO ligand and a larger solvent accessibility in P. stutzeri attributed to the lack of tyrosine residue in its N-terminus. In P. aeruginosa cd1 NIR Tyr10 is H-bonded to the NO, afforded by domain swapping. These findings support an earlier suggestion that the large difference in the c- to d1-heme electron transfer rates in the two enzymes is associated with the open conformation of the d1-heme pocket