Electron paramagnetic resonance studies on Pseudomonas nitrosyl nitrite reductase. Evidence for multiple species in the electron paramagnetic resonance spectra of nitrosyl haemoproteins

Abstract

The EPR spectra of reduced 14NO- and 15NO-bound Pseudomonas nitrite reductase were investigated at pH 5.8 and 8.0 in 4 buffer systems. At pH 8.0, absorption spectra indicated that only the heme d1 was NO-bound, but, although quantification of the EPR signals in all cases accounted for NO bound to the heme d1 in both subunits of the enzyme, the precise form of the signals varied with buffer and temperature. A rhombic species, with gx = 2.07, gz = 2.01 and gy = 1.96, represented in the low-temperature spectra seen in all the buffers was converted at high temperatures (approximately 200.degree. K) into a form showing a reduced anisotropy. Hyperfine splitting on the gz component of this rhombic signal indicated a nitrogen atom trans to NO and it is proposed that histidine provides the endogenous axial ligand for heme d1. At pH 5.8, absorption spectra indicated NO binding to both hemes c and d1 and EPR quantifications accounted for NO-bound hemes c and d1 in both enzyme subunits. The EPR spectra at pH 5.8 were generally similar to those at pH 8.0 with respect to g-values and hyperfine coupling constants, but were broader with less well defined hyperfine splittings. As at pH 8, rhombic signals present in spectra at low temperatures were converted to less anisotropic forms at high temperatures. The results are discussed in relation to work on model nitrosyl-protoheme complexes. No EPR signal was observed from oxidized NO-bound Pseudomonas nitrite reductase at pH 6.0, over the temperature range 6-100.degree. K.