Infrared spectroscopy of carbonmonoxide ligation to the coelomic hemoglobins of glycera dibranchiata

Glycera dibranchiata is a marine annelid which possesses both a high molecular weight hemoglobin fraction and a monomer hemoglobin fraction of molecular weight 15,590D [1–8]. NMR studies along with companion isoelectric focusing and column chromatography work have established the extensive microheterogeneity of the monomer hemoglobin fraction [9]. However, to date, it has been impossible to successfully obtain proton nmr spectra of the high molecular weight fraction. In part, thisis due to its ready tendency to aggregate into a gel state. Subsequently, infrared spectroscopy has been used to probe the heme ligand binding site in both of the Glycera hemoglibin fractions. Experimental Infrared spectra of Glycera hemoglobin fractions were obtained using CaF cells. Isolation of Glycera hemoglobin fractions has been previously detailed [9], and the proteins were handled in 0.1 M phosphate buffer (Mallinkrodt), pH 6.8. 2H2O (99.8%, Merck) was selected as the buffer solvent to insure an infrated window between 2000 and 1920 cm−1 infrared spectra were obtained either on a Nicolet 6000 FTIR or a Perkin Elmer 621 grating spectrometer. Results and Discussion Figure 1 reveals the existence of at least three CO stretching frequencies, indicating multiple heme bound carbon monoxide coordination sites in the unseparated hemoglobin mixture and in the separated higher molecular weight fraction. The separated monomer fraction exhibits only a single CO infrared frequency at 1970 cm−1, identical to this frequency in ferrous porphyrinCO [10–12]. This is interesting in view of the fact that X-ray crystallography [3] indicates that at least one of the monomer component hemoglobins lacks the ‘ubiquitous’ distal histidine (E-7). Because the essential ‘myoglobin fold’ structure is preserved in the Glycera monomer along with a substantial degree of amino acid conservation in the primary sequence of the heme pocket residues [6], this data suggests the influence which a distal residue exerts on the spectroscopic properties of heme bound CO. Likewise, the carbon-13 NMR frequency of heme bound 13CO in the Glycera dibranchiata monomer fraction has been reported to be identical to imidazole—ferrous porphyrin—13CO molecules [13, 14]. This data suggests an interesting correlation between infrared stretching frequencies for heme bound 12CO or 13CO and chemical shifts for the same molecules with heme bound 13CO. The emperical result is shown in Fig. 2. That figure further suggests that the type of distal ligand in heme proteins may be ascertained either by NMR 13CO chemical shift, or by infrated CO frequency. It is also to be noted that among the proteins which exhibit νCO near 1950 cm−1 there are slight differences. This is obvious for rabbit α chain νCO and β chain νCO and between human hemoglobin νCO and whale myoglobin νCO. Similar differences occur in the C-13 NMR spectra. It is likely that these differences can be attributed to the degree of distal histidine interaction with heme bound CO. The correlation is one where enhanced interaction reflects downfield 13CO shifts and infrared νCO bands at lower wavenumbers. Therefore, the correlation of Fig. 2 reflects the distal ligand interaction with heme bound CO as well as the nature of the distal ligand. Additional pieces of evidence for this view originate with elephant myoglobin [15]. This is a unique mammalian myoglobin in that the distal histidine is replaced by glutamine in the primary sequence [15]. Although no exact values of 13CO chemical shift or νCO have yet been published, current observations indicate that νCO lies at lower wavenumbers than νCO for myoglobin [16]. Similarly, the unreported 13CO chemical shift appears further downfield than in mammalian hemoglobins [17]. Thus, until specific values are published, what can be said is that elephant myoglobin conforms to the correlation of Fig. 2, in general terms. What can be concluded is that the position of elephant myoglobin–CO in the correlation indicates that the distal residue-CO interaction in this protein is enhanced over normal mammalian hemoglobins and myoglobins. That is, there seem to be ‘better’ distal ligands than the ubiquitous histidine E-7. A further conclusion is that 13CO chemical shifts or infrared frequencies of CO-protein may be a quick, useful analytical technique for characterizing the distal amino acid in unsequenced b-type heme proteins.