RESONANCE RAMAN-SPECTROSCOPIC STUDIES OF LIGATED MUTANT MYOGLOBINS

Ligand binding (CO and N-3(-)) to wild-type porcine myoglobin and to several mutant forms, expressed and purified from E. coli cells, has been studied using Raman spectroscopy. The v(Fe-CO) stretching vibration in Mb(II)CO has been compared for the wild-type and mutant proteins. This gives a broad band consisting of five components, indicating five possible configurations of the bound CO. The distal pocket mutants show large variations in bandshape, the major component occurring at progressively lower wavenumber in the order: wild-type (WT) > E11 Val --> Thr (VT) > E7 His --> Val (HV) > the double mutant VT/HV (M2). Changes observed in the Raman band assigned to the azide bending mode in Mb(III)N(3) have been interpreted in terms of resonance structures involving two forms of azide binding. Repulsion between the bound azide ligand and the OH group of the adjacent thr residue in the VT mutant, and a shorter Fe-N(his) bond in the proximal mutant Ser --> Leu (F7), both affect this bonding. In the wild-type protein (WT), hydroxymetmyoglobin exists in a spin-state equilibrium which, at room temperature, is predominantly high-spin. In the F7 mutant this equilibrium is shifted in favour of the low-spin form. A low-spin iron species also exists in the aquometmyoglobin form of this mutant.