LIGAND-BINDING AND SLOW STRUCTURAL-CHANGES IN CHLOROCRUORIN FROM SPIROGRAPHIS-SPALLANZANII

Chlorocruorin is a cooperative respiratory pigment found in the blood of polychaete worms; its prosthetic group is a derivative of the iron protoporphyrin IX, in which the vinyl group at position 2 is substitut ed by a formyl group. The quaternary structure of chlorocruorins is co mplex: myoglobin-like subunits are grouped in tetramers and tetramers in dodecamers; 12 dodecamers are assembled in the 3500-kDa particle. C hlorocruorin from Spirographis spallanzanii displays the following ove rall functional properties: (i) the oxygen affinity is lower than in h uman hemoglobin, while that of CO is similar if not higher; (ii) the r ates of combination with oxygen and carbon monoxide are low; and (iii) the off rate of oxygen is comparable to that of human hemoglobin, whi le the off rate of CO is 10 times smaller. When CO is partially photol yzed with a long and powerful light flash (70 mus), rebinding is bipha sic as in mammalian hemoglobins; however, the slowest rate is faster t han that observed by stopped flow, suggesting that the unliganded prot ein decays from the liganded high affinity state (R) to an intermediat e state before reaching the low affinity (T) state. Oxygen binding was followed by stopped-flow and flash photolysis. While partial photolys is yields a fast, second-order time course, stopped-flow experiments y ield slow, biphasic, and non-second-order time courses. This pattern o f reactivity was attributed to a slow conformational transition(s) whi ch is (are) rare limiting with oxygen, but not with CO. Photolysis wit h a short (9-ns) laser pulse shows first-order rebinding processes, at tributed (by analogy to hemoglobins) to the recombination of the ligan d from inside the protein matrix (geminate rebinding). In the bimolecu lar processes observed by laser photolysis, the rebinding rate charact eristic of the intermediate state is not observed; this was attributed to the relatively slow decay from the R to the intermediate state (X) . These experiments indicate that (some of) the allosteric transition( s) in Spirographis chlorocruorin is (are) slow and populate(s) long-li ved intermediates with reactivities different from those of the T and R states.