2 ENZYMES WITH A COMMON FUNCTION BUT DIFFERENT HEME LIGANDS IN THE FORMS AS ISOLATED - OPTICAL AND MAGNETIC-PROPERTIES OF THE HEME GROUPS IN THE OXIDIZED FORMS OF NITRITE REDUCTASE, CYTOCHROME CD(1), FROM PSEUDOMONAS-STUTZERI AND THIOSPHAERA-PANTOTROPHA

It is shown that, in the oxidized state, heme c of Pseudomonas stutzer i (ZoBell strain) cytochrome cd(1) has histidine-methionine ligation a s observed for cytochrome cd(1) from Pseudomonas aeruginosa [Sutherlan d, J., Greenwood, C., Peterson, J., and Thomson, A. J. (1986) Biochem. J. 233, 893-898]. However, the X-ray structure of Thiosphaera pantotr opha cytochrome cd(1) reveals bis-histidine ligation for heme c. It is confirmed by EPR and near-infrared (NIR) MCD measurements that the bi s-histidine coordination remains unaltered in the solution phase. Henc e, the difference between the heme c ligation states defines two disti nct classes of oxidized cytochromes cd(1) as isolated. A weak feature in the T. pantotropha NIR MCD at 1900 nm suggests that a small populat ion of heme c has histidine-methionine coordination. The Ligation stat e of heme d(1) cannot be defined with the same level of confidence, be cause the porphyrin-to-Fe(III) charge-transfer (CT) bands are less wel l characterized for this class of partially reduced porphyrin ring. Ho wever, variable temperature absorption and MCD spectra show that, in t he T. pantotropha enzyme, heme d(1) exists in a thermal low-spin/high- spin mixture with the low-spin as the ground state, whereas in P. stut zeri cytochrome cd(1), and d(1) heme is low-spin at all temperatures. A weak band, assigned as the heme d(1) porphyrin-pi(a(1u),a(2u))-to-fe rric(d) charge-transfer transition has been identified for the first t ime at 2170 nm. Its magnetic properties show the heme d(1) to have an unusual (d(xz,yz))(4)(d(xy))(1) electronic ground state as is found fo r low-spin Fe(III) chlorins [Cheesman, M. R., and Walker, F. A. (1996) J. Am. Chem. Sec. 118, 7373-7380]. It is proposed that the localizati on of the Fe(III) unpaired d-electron in an orbital lying in the heme plane may decrease the affinity of the ee(III) heme for unsaturated li gands such as NO. Although heme d(1) in the enzymes from P. stutzeri a nd T. pantotropha shows different temperature-dependent spin propertie s, the positions of the low-spin Fe(III) alpha-absorption band, at sim ilar to 640 nm, are very similar to those observed for cytochromes cd( 1) from eight other sources, suggesting that all have similar strength fields from the axial ligands and, hence, that all have the same coor dination, namely histidine-tyrosine or possibly histidine-hydroxide at the heme.