Distal interactions in the cyanide complex of ferric Chlamydomonas hemoglobin

Chlamydomonas hemoglobin is expressed in chloroplast during active photosyn thesis. Its heme pocket has an unusual structure that undergoes substantial changes when exogenous ligands bind the heme iron. In the ferrous state of the heme, oxygen binds with high affinity and is stabilized by interaction s from E7-glutamine and BIO-tyrosine. In the present study, we have examine d cyanide binding to the ferric henle by resonance Raman spectroscopy. The frequency of the Fe-CN stretching mode in the wild-type protein was assigne d at 440 cm(-1), which is significantly lower than that observed in other g lobins. Another cyanide isotope sensitive mode located at 315 cm(-1) is ten tatively assigned as an Fe(III)-His stretching mode in the six-coordinate C N adduct. To determine the sensitivity of the Fe-CN stretching mode to the interactions in the distal pocket, we also studied three distal pocket muta nts. The frequency of the Fe-CN stretching mode in the cyanide complex of t he Lys87Ala (E10) mutant was nearly identical to that of the wild-type prot ein but it increased to 452 cm(-1) for the Gln4Gly (E7) mutant. In the Tyr6 3Leu (B10) mutant, the frequency of the Fe-CN stretching mode decreased by 5 cm(-1) relative to that in the wild type. On the basis of the low frequen cy for the Fe-CN stretching mode in the wild-type protein and its smaller t han expected cyanide isotope shift (4 cm(-1) compared to 11 cm(-l) expected for a two-body Fe-CN oscillator) we propose a highly bent Fe-C-N structure in Chlamydomonas hemoglobin, in sharp contrast to the widely accepted line ar cyanide structure in most vertebrate globins. The occurrence of a bent c yanide structure in Chlamydomonas hemoglobin is likely caused by a congeste d distal cavity resulting in a strong steric interaction between the E7-glu tamine residue and the heme-bound cyanide.