Transient ligand docking sites in Cerebratulus lacteus mini-hemoglobin

Abstract

The monomeric hemoglobin of the nemertean worm Cerebratulus lacteus functions as an oxygen storage protein to maintain neural activity under hypoxic conditions. It shares a large, apolar matrix tunnel with other small hemoglobins, which has been implicated as a potential ligand migration pathway. Here we explore ligand migration and binding within the distal heme pocket, to which the tunnel provides access to ligands from the outside. FTIR/TDS experiments performed at cryogenic temperatures reveal the presence of three transient ligand docking sites within the distal pocket, the primary docking site B on top of pyrrole C and secondary sites C and D. Site C is assigned to a cavity adjacent to the distal portion of the heme pocket, surrounded by the B and E helices. It has an opening to the apolar tunnel and is expected to be on the pathway for ligand entry and exit, whereas site D, circumscribed by TyrB10, GlnE7, and the CD corner, most likely is located on a side pathway of ligand migration. Flash photolysis experiments at ambient temperatures indicate that the rate-limiting step for ligand binding to CerHb is migration through the apolar channel to site C. Movement from C to B and iron-ligand bond formation involve low energy barriers and thus are very rapid processes in the wt protein.

The Apolar Channel in Cerebratulus lacteus Hemoglobin Is the Route for O2 Entry and Exit

“…Some of these rate parameters were reported in previous publications (21,32). We constructed the Val-44 mutant to complete the series of size variants at the E7 gate position and re-measured all the O 2 and CO binding parameters for direct comparison with the same replacements in sperm whale Mb, which uses the E7 gate for ligand entry and exit (Fig.…”

“…Thus, all bound ligands are experiencing a partial negative charge from the nonbonded electrons of the O-atom of Tyr-11(B10), which accounts for the moderate O 2 affinity (Table 2) and high stretching frequency of bound CO (ϳ1980 cm Ϫ1 , Fig. 8) (21,32) for WT CerHb and all five position 55 mutants. A complete discussion of the key role of Thr-48(E11) in regulating O 2 binding in CerHb is given in Pesce et al (32).…”

“…Mutants-We have shown previously that, when photodissociated at low temperatures, CO occupies several positions (denoted as B, C, and D) within the distal pocket of CerHb before migrating into the apolar channel (21). On the basis of the effects of distal pocket mutations, the B state was assigned to a position underneath Phe-10(B9) and Tyr-11(B10), directly above pyrrole C. The C state was identified as a position located further into the protein interior beneath Val-7(B6) and above the vinyl group of pyrrole B, just at the beginning of the apolar channel (21) (Fig.…”

“…On the basis of the effects of distal pocket mutations, the B state was assigned to a position underneath Phe-10(B9) and Tyr-11(B10), directly above pyrrole C. The C state was identified as a position located further into the protein interior beneath Val-7(B6) and above the vinyl group of pyrrole B, just at the beginning of the apolar channel (21) (Fig. 9).…”

Ligand Migration and Binding in the Dimeric Hemoglobin ofScapharca inaequivalvis,

“…For example, an exceptionally large internal volume of ∼300 Å 3 exists in neuroglobin, which allows the entire heme prosthetic group to slide deeper into the protein so as to resolve steric conflicts between the exogenous ligand and the distal histidine (97). In the mini-hemoglobin of Cerebratulus lacteus, a channel in the protein matrix provides a ligand migration pathway (81). In Mb, the Xe4 and Xe1 cavities serve as transient ligand storage sites (18,20,28,30,69,93,98).…”