Ultramicroscopy 9 (1982) 3 1-36 North-Holland Publishing Company 31 zyxwvutsrq STEREOCHEMISTRY OF IRON IN DEOXYHAEMOGLOBIN * M.F. PERUTZ MRC Laboratory of Molecular Biology, Cambridge CB2 2QH, UK S. SAMAR HASNAIN and P.J. DUKE Daresbury Laboratory, Warrington WA4 4AD, UK and J.L. SESSLER and J.E. HAHN Department of Chemistry, Stanford University, Stanford, California 94305, USA Received 18 March 1982 The EXAFS of human deoxyhaemoglobin closely resembles that of a synthetic model in which the displacement of the iron from the mean porphyrin planeis 0.426--~0.004 A, similar to the displacement of 0.56*0.03 A found by single crystal X-ray analysis of deoxyhaemoglobin. We find the same Fe-N of 2.0620.01 A distance as Eisenberger et al., but show that the displacement of the iron from the nitrogen plane cannot be calculated from that distance. X-ray crystallography has shown that in de- oxyhaemoglobin the iron atoms are displaced by 0.56 * 0.03 A from the mean porphyrin plane, whereas in liganded haemoglobins they lie either in or close to that plane [1,2]. Perutz [3] proposed this shift as one principal factor responsible for haem-haem interaction. Eisenberger et al. used extended X-ray absorption fine structure (EXAFS) to measure the Fe-N distance in deoxyhaemoglo- bin and concluded that the irons lie only 0.2’$ A from the plane of the porphyrin nitrogens and that the mechanism of Perutz is therefore invalid [4]. We have now compared the EXAFS of de- oxyhaemoglobin with that of the ferrous “picket fence” 2-methylimidazole complex in which the displacement of the iron from the plane of the porphyrin nitrogens is known to be 0.399 * 0.004 A and from the mean porphyrin plane 0.426 f 0.004A [5]. The two EXAFS spectra are very * This paper has first appeared in Nature and is included in this issue with kind permission of the Editor of Nature. similar, consistent with similar displacements of the irons. We find the same Fe-N distance of 2.06 * 0.01 A in deoxyhaemoglobin as Eisenberger et al., but show that the displacement of the iron cannot be calculated from that distance. The samples were prepared as follows. A 15 mM solution of human deoxyhaemoglobin was prepared from a red cell lysate by pressure dialysis followed by deoxygenation and addition of neutral Na,S,O, in an oxygen-free glove box. A drop of this solution was mounted in a hole 5 mm wide in a 1 mm thick copper strip, sealed between thin mylar films and kept under nitrogen before use. The crystalline picket fence complex (2-methyl-im- idazole-meso-tetra(c, (Y, (Y, cu-O-pivamidophenyl)- porphyrinatoiron-(II)-ethanol [Fe’+(T piv PP)-(2- MeIm) . EtOH] was prepared in oxygen-free con- ditions as described in ref. [5]. In the first of the two independent EXAFS experiments, 2 mm wide thin-walled glass capillaries were filled with the sample and sealed with bees wax. In the second, the complex was diluted by mixing with boron 0304-3991/82/0000-0000/$02.75 Q 1982 North-Holland