Two 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

• Published in: Biochemistry (Easton) 1997-12, Vol.36 (51), p.16267-16276
• Main Authors: Cheesman, Myles R, Ferguson, Stuart J, Moir, James W. B, Richardson, David J, Zumft, Walter G, Thomson, Andrew J
• Format: Article
• Language: English
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi971677a

It is shown that, in the oxidized state, heme c of Pseudomonas stutzeri (ZoBell strain) cytochrome cd 1 has histidine-methionine ligation as observed for cytochrome cd 1 from Pseudomonas aeruginosa [Sutherland, J., Greenwood, C., Peterson, J., and Thomson, A. J. (1986) Biochem. J. 233, 893−898]. However, the X-ray structure of Thiosphaera pantotropha cytochrome cd 1 reveals bis-histidine ligation for heme c. It is confirmed by EPR and near-infrared (NIR) MCD measurements that the bis-histidine coordination remains unaltered in the solution phase. Hence, the difference between the heme c ligation states defines two distinct classes of oxidized cytochromes cd 1 as isolated. A weak feature in the T. pantotropha NIR MCD at 1900 nm suggests that a small population of heme c has histidine-methionine coordination. The ligation state of heme d 1 cannot be defined with the same level of confidence, because the porphyrin-to-Fe(III) charge-transfer (CT) bands are less well characterized for this class of partially reduced porphyrin ring. However, variable temperature absorption and MCD spectra show that, in the 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. stutzeri cytochrome cd 1, and d 1 heme is low-spin at all temperatures. A weak band, assigned as the heme d 1 porphyrin-π(a1u,a2u)-to-ferric(d) charge-transfer transition has been identified for the first time at 2170 nm. Its magnetic properties show the heme d 1 to have an unusual (d x z ,y z )4(d x y )1 electronic ground state as is found for low-spin Fe(III) chlorins [Cheesman, M. R., and Walker, F. A. (1996) J. Am. Chem. Soc. 118, 7373−7380]. It is proposed that the localization of the Fe(III) unpaired d-electron in an orbital lying in the heme plane may decrease the affinity of the Fe(III) heme for unsaturated ligands such as NO. Although heme d 1 in the enzymes from P. stutzeri and T. pantotropha shows different temperature-dependent spin properties, the positions of the low-spin Fe(III) α-absorption band, at ∼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 coordination, namely histidine-tyrosine or possibly histidine-hydroxide at the heme.