Distal interactions in the cyanide complex of ferric Chlamydomonas hemoglobin

Chlamydomonas hemoglobin is expressed in chloroplast during active photosynthesis. Its heme pocket has an unusual structure that undergoes substantial changes when exogenous ligands bind the herne iron. In the ferrous state of the heme, oxygen binds with high affinity and is stabilized by interactions from E7-glutamine and B10-tyrosine. In the present study, we have examined cyanide binding to the ferric heme by resonance Raman spectroscopy. The frequency of the Fe-CN stretching mode in the wild-type protein was assigned at 440 cm^-1, which is significantly lower than that observed in other globins. Another cyanide isotope sensitive mode located at 315 cm^-1 is tentatively assigned as an Fe(III)-His stretching mode in the six-coordinate CN adduct. To determine the sensitivity of the Fe-CN stretching mode to the interactions in the distal pocket, we also studied three distal pocket mutants. The frequency of the Fe-CN stretching mode in the cyanide complex of the Lys87Ala (E10) mutant was nearly identical to that of the wild-type protein but it increased to 452 cm^-1, for the Gln84Gly (E7) mutant. In the Tyr63Leu (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 frequency for the Fe-CN stretching mode in the wild-type protein and its smaller than expected cyanide isotope shift (4 cm^-1 compared to 11 cm^-1 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 linear cyanide structure in most vertebrate globins. The occurrence of a bent cyanide structure in Chlamydomonas hemoglobin is likely caused by a congested distal cavity resulting in a strong steric interaction between the E7-glutamine residue and the heme-bound cyanide.