BIS-METHIONINE LIGATION TO HEME IRON IN MUTANTS OF CYTOCHROME B(562) .1. SPECTROSCOPIC AND ELECTROCHEMICAL CHARACTERIZATION OF THE ELECTRONIC-PROPERTIES
Pd. Barker et al., BIS-METHIONINE LIGATION TO HEME IRON IN MUTANTS OF CYTOCHROME B(562) .1. SPECTROSCOPIC AND ELECTROCHEMICAL CHARACTERIZATION OF THE ELECTRONIC-PROPERTIES, Biochemistry, 35(42), 1996, pp. 13618-13626
We have generated mutants of cytochrome b(562) in which the histidine
ligand to the heme iron (His102) has been replaced by a methionine, Th
e resulting proteins can have bis-methionine coordination to the heme
iron, but the stability of this arrangement is dependent on oxidation
stare and solution pH. We have used optical, MCD, and EPR spectroscopi
es to study the nature of the heme coordination environment under a va
riety of conditions. Optical spectra of the reduced state of the singl
e variant, H102M, are consistent with bis-methionine ligation. In its
oxidized state, this protein is high-spin under all conditions studied
, and the spectroscopic properties are consistent with only one of the
methionine ligands being coordinated. We cannot identify what, if any
thing, provides the other axial ligand. A double variant, R98C/H102M (
in which the heme is covalently attached to the protein through a c-ty
pe thioether linkage), is also bis-methionine coordinated in the ferro
us state, but has significantly different properties in the oxidized s
tate. With a pK(a) of 7.1 at 20 degrees C, the protein converts from a
low-spin, 6-coordinate heme protein at low pH, to a high-spin species
, similar to the high-spin species observed for the single variant. Ou
r spectroscopic data prove that the low-spin species is bis-methionine
coordinated. The reduction potential of this bis-methionine species h
as been measured using direct electrochemical techniques and is +440 m
V at pH 4.8. The electrochemistry of these proteins is complicated by
coupled coordination-state changes. Proof that the ferrous state is bi
s-methionine coordinated is provided by NMR results presented in the f
ollowing paper.