A Double Mutant of Sperm Whale Myoglobin Mimics the Structure and Function of Elephant Myoglobin

Abstract

The functional, spectral, and structural properties of elephant myoglobin and the L29F/H64Q mutant of sperm whale myoglobin have been compared in detail by conventional kinetic techniques, infrared and resonance Raman spectroscopy, 1H NMR, and x-ray crystallography. There is a striking correspondence between the properties of the naturally occurring elephant protein and those of the sperm whale double mutant, both of which are quite distinct from those of native sperm whale myoglobin and the single H64Q mutant. These results and the recent crystal structure determination by Bisig et al. (Bisig, D. A., Di Iorio, E. E., Diederichs, K., Winterhalter, K. H., and Piontek, K.(1995) J. Biol. Chem. 270, 20754-20762) confirm that a Phe residue is present at position 29 (B10) in elephant myoglobin, and not a Leu residue as is reported in the published amino acid sequence. The single Gln64(E7) substitution lowers oxygen affinity ∼5-fold and increases the rate of autooxidation 3-fold. These unfavorable effects are reversed by the Phe29(B10) replacement in both elephant myoglobin and the sperm whale double mutant. The latter, genetically engineered protein was originally constructed to be a blood substitute prototype with moderately low O2 affinity, large rate constants, and increased resistance to autooxidation. Thus, the same distal pocket combination that we designed rationally on the basis of proposed mechanisms for ligand binding and autooxidation is also found in nature. The functional, spectral, and structural properties of elephant myoglobin and the L29F/H64Q mutant of sperm whale myoglobin have been compared in detail by conventional kinetic techniques, infrared and resonance Raman spectroscopy, 1H NMR, and x-ray crystallography. There is a striking correspondence between the properties of the naturally occurring elephant protein and those of the sperm whale double mutant, both of which are quite distinct from those of native sperm whale myoglobin and the single H64Q mutant. These results and the recent crystal structure determination by Bisig et al. (Bisig, D. A., Di Iorio, E. E., Diederichs, K., Winterhalter, K. H., and Piontek, K.(1995) J. Biol. Chem. 270, 20754-20762) confirm that a Phe residue is present at position 29 (B10) in elephant myoglobin, and not a Leu residue as is reported in the published amino acid sequence. The single Gln64(E7) substitution lowers oxygen affinity ∼5-fold and increases the rate of autooxidation 3-fold. These unfavorable effects are reversed by the Phe29(B10) replacement in both elephant myoglobin and the sperm whale double mutant. The latter, genetically engineered protein was originally constructed to be a blood substitute prototype with moderately low O2 affinity, large rate constants, and increased resistance to autooxidation. Thus, the same distal pocket combination that we designed rationally on the basis of proposed mechanisms for ligand binding and autooxidation is also found in nature. INTRODUCTIONMyoglobin is a small, 16-18-kDa, globular hemeprotein which stores molecular oxygen in muscle cells. Because of the similarity of their tertiary structures, myoglobin is frequently used as a model for the α and β subunits of tetrameric hemoglobin. The key physiological properties of myoglobin are its high affinity for oxygen and low rate of spontaneous oxidation to the inactive ferric form (Antonini and Brunori, 1971). Studies of naturally occurring and mutant hemeproteins have shown that ligand affinity and autooxidation are affected markedly by the chemical nature of the amino acids in the heme pocket. In order to understand these functional properties, the molecular structures of a large number of myoglobins and hemoglobins have been determined by x-ray crystallography and solution NMR studies. These studies have been pursued along two lines. Traditionally, naturally occurring genetic or species variants with distinctive functional properties have been investigated (i.e. Bolognesi et al.(1989), Steigmann and Weber(1979), Vainshtein et al.(1978), Yu et al.(1990), Qin and La Mar (1992), and Vyas et al.(1993)). More recently, site-directed mutagenesis has been used either to test structural hypotheses (Olson et al., 1988; Braunstein et al., 1988; Carver et al., 1990; Ikeda-Saito et al., 1991; Balasubramanian et al., 1993; Li et al., 1994; Ling et al., 1994; Huang and Boxer, 1994) or to explore systematically the physiological roles of individual residues (Rohlfs et al., 1990; Egeberg et al., 1990; Smerdon et al., 1991, 1993; Carver et al., 1991, 1992; Quillin et al., 1993, 1995; Lai et al., 1995). A review of the mutagenesis literature is given in Springer et al.(1994).Dene et al.(1980) sequenced Asian elephant myoglobin and reported that it contains a Gln at position 64 (E7) rather than a His, which is found at this position in all other mammalian myoglobins. This replacement appeared to be neutral since the O2 affinity and autooxidation rate of elephant myoglobin are very similar to those of pig, human, and, sperm whale Mb 1The abbreviations used are: MbdeoxymyoglobinMbCOcarbonmonoxy et al., Springer et al., et that in elephant myoglobin, of Gln64(E7) a with oxygen which is in to that with in all other mammalian myoglobins. this was by the Gln in sperm whale myoglobin, the results that this substitution is not The H64Q a in in a Studies at H64Q sperm whale myoglobin not the functional and properties of elephant myoglobin have been pursued by two La Mar and et al., 1990; Vyas et al., have high 1H NMR studies of both and of elephant Mb and compared the results to native sperm whale myoglobin as a The in to for the distal are also which that a is in with ligand in the elephant and et al., 1992; et al., 1992; Springer et al., 1994; Li et al., 1994) have a of at position 29 (B10) in sperm whale of Phe for in sperm whale myoglobin to a in O2 affinity and in autooxidation rate et al., The crystal structure of this mutant that of the with The is by with the of the for the in affinity shown by the mutant. The large also from the distal pocket of the form of the mutant. structure of has been determined to by Quillin as a of The of the and Phe29(B10) are very similar to those reported for the and structures of this mutant. A of the structure is the of distal pocket to the large of the In native and sperm whale a is found to of and to the heme et al., The and effects also of the and et al., to a blood substitute and et al., 1993; 1994) constructed a double mutant in which Phe was at the 29 position to autooxidation by from the and Gln was at the 64 position to O2 affinity by to the it was that this was to in elephant Mb by Yu et the published for the elephant protein a Leu at the position et al., this and test the of Yu et al.(1990), we have compared in detail the spectral, and functional properties of elephant myoglobin and the L29F/H64Q sperm whale mutant. for O2 and binding and autooxidation determined by conventional kinetic of the of and L29F/H64Q sperm whale myoglobins compared with that of elephant Raman used to the and for the same of and native The structure of the of the form of the sperm whale double mutant was determined by solution 1H NMR and compared with that of the single and elephant the structure of L29F/H64Q sperm whale was determined by x-ray crystallography for with the determined by NMR and with the crystal structure of native elephant by Bisig et and and of the mutagenesis of sperm whale myoglobin was given by Carver et the of and was for and The of these was The of mutagenesis was used to the mutant. The double sperm whale mutant was constructed the by Springer et to the H64Q single mutant. The was sequenced by the as by and a the mutant the and as by Springer and also Lai et The myoglobins to to and in and for O2 and binding determined conventional and as in detail by et for O2 and binding as the of the rate the by et sperm whale myoglobin in E. has been shown to be with native sperm whale myoglobin in of tertiary structure and kinetic properties et al., Thus, the for sperm whale myoglobin also to the native and Raman the infrared of the solution was to a to a The of by a and was with the was at in the a with a to for all and the The for by of the and the resonance Raman the of the by with of The by oxidation with and of and and by in a to in the of and in the of was as by the of from the of the The resonance Raman a with a with as the was by the of a The was and the was NMR for 1H NMR by to the by to the and in The solution was heme in was for the the 1H NMR on the the was by the for the the as in detail and La the of the to a the and was from the of the of the in the et al., The and and et al., and the same and in the same as in detail et al., 1993, 1994; Qin and La determined as in detail and La et al., 1993; Qin et al., for the of the heme used as to for the that the molecular or from crystal by the and are and and is the chemical to is the in the and or for are not by the et al., is the of amino acid for et al., and is the of the on the by the model and the in was and or to all to both the and as in detail et al., by position of the was by the of the from the heme and to and as in detail et al., The of Gln64(E7) was by the with that and the for the as in detail et al., the was determined by the that both the and as et al., et al., to the for was a with and is The molecular was on a for the structure the was in solution the by et The form of the double mutant in the with with a as et al., The a The was to a of with was of used as the model in the of the double mutant structure Quillin et al. the et al., The Gln64(E7) and Phe29(B10) the the molecular and by the and for the myoglobins and of and the to with of for the L29F/H64Q structure have been in the and rate and for ligand binding to and L29F/H64Q sperm whale and elephant myoglobins are in in this the autooxidation rate The H64Q substitution a in the rate of O2 to of between residue 64 (E7) and the ligand et al., 1994; Quillin et al., The is a in and Raman of to sperm whale and native elephant myoglobins are shown in sperm whale myoglobin two in the of its a at and a at for a review Li et The a to a single at The position for the H64Q mutant is the same as that of sperm whale myoglobin, is a of in the and L29F/H64Q sperm whale and native elephant myoglobin at infrared of at Raman of the and in the of the and are shown in a review of these in hemoglobins and myoglobins Yu and The for the and with and Ling et al. The single in from for to and the single H64Q replacement a to both are as in the double mutant and elephant myoglobin, is results are for the The a in the H64Q replacement a the for the double mutant and elephant myoglobin are to that of sperm whale the results in that elephant myoglobin contains a Phe29(B10) residue which with in a with that in the L29F/H64Q double mutant of sperm whale These also the of the and to amino acid in the distal resonance Raman of at resonance Raman of at of 1H NMR for of by a single of of a molecular a a of which for of the These two of from heme which is a of not The of the of to with the the of the NMR to those to the and L29F/H64Q in are shown in The for these a similar not that of sperm whale myoglobin and other et al., 1993; Qin et al., The as for other in the of the ferric The for the heme and residues has been and in detail for both sperm whale and and La Qin and La 1992; et al., 1993; Qin et al., NMR for L29F/H64Q are shown to and the two residues Gln64(E7) and of the 1H NMR at at in at at in at at in in of in at to of in at of in at at a rate of 29 1H NMR of in is shown in and are found in the low by to the in shown in of these is in a in which a the that is in the same in The of in shown in the the for of of and of and the of and the of of for the heme in all as by Qin and La of the and and the for are by and and La 1992; Qin et al., is by and its to the and are by the for and the of to A for with to the and Phe which to Gln64(E7) and and and La Mar et al., for and to at with to and the of studies on the two single chemical for the heme model of L29F/H64Q as as on the two single are in to in et of of of The to as in et in to the of and of The et al., in to the of of and of of and of have not been of these the and of have been in a The and of the double mutant and and that are from those for and La et al., Thus, the structure on the of the heme is in all the the distal residues also and and very similar to those for sperm whale The structural to the and of Gln64(E7) and Phe29(B10) in is in as two and which are by a between and is to the to The in to Gln64(E7) and These of Gln64(E7) at a of from the The is to the to The Gln64(E7) in the single H64Q mutant was in a similar of of the of the in at for the Phe29(B10) and The are are by to to and the of the of at the of Phe29(B10) and The by a to to and The that the is in the other the for the at a of both and L29F/H64Q two and with for a Phe that from Phe29(B10) The properties of for of Phe29(B10) in both with the for in the mutant and in the L29F/H64Q double mutant. Phe29(B10) in L29F/H64Q not to heme to and as as a to Gln64(E7) These are from the crystal structures of both the single et al., and double L29F/H64Q and are similar to those by the residue in native and and Quillin et al., These the distal pocket residues are in in a distal heme pocket structure of and and and of the for the and for the and sperm whale myoglobin are in The between and are and the The results from a determination for are for the basis of studies of et that the by and and that these the in the The of in the to in the and the This is with the in the low of in a of the from to of and of are in L29F/H64Q than in the protein and very similar to those found in In the mutant, these have been shown to from of the the et al., of the by between and for the of Gln64(E7) in L29F/H64Q was determined by for which the of the single and double mutant and the for the on the as the was with the for was at the for the as in detail by Qin et al. The of Gln64(E7) in the double mutant is with that determined for the single mutant and from the a which is with that by this determined by 1H NMR from that found in the two crystal structures, a to the was not and used as to the of Phe29(B10) in L29F/H64Q shown in and a of the a of The for is at is The was found to be to also with those from and The of the affected from for the Phe29(B10) as a of and for Phe in the sperm whale crystal with the the heme by the The of and are with the from as shown by of L29F/H64Q of the of L29F/H64Q myoglobin is shown in A of the of the to the ligand with those in the structures of the single is in The not and in the double mutant which are with those in the crystal structures of the single The ligand is in with both of Gln64(E7) and the of the of the functional and effects shown in There is also a correspondence between the from the and the in the solution NMR with These results the NMR and that the structures of the distal in solution are similar not those in structures of L29F/H64Q sperm whale and elephant myoglobins. for the distal pocket of L29F/H64Q sperm whale The of is in a position to with the The of is in a position to with the ligand and a position similar to that of a in which is found in all mammalian myoglobins. of the structures of the distal of L29F/H64Q sperm whale myoglobin and elephant myoglobin Bisig et The of the are very similar in both which to their similar functional The with Bisig et have determined the high crystal structure of native Asian elephant shown in a Phe residue is present at the 29 position in the elephant and the of the and are with those in the sperm whale double of the Mb myoglobin has in to structure with in This protein the same O2 affinity and resistance to autooxidation as the mammalian myoglobins from pig, and sperm whale a Gln is present at the position Springer et al., the mutagenesis of Springer et al.(1989), it was that this H64Q substitution was neutral and that the O2 to the same as the in as shown in the single H64Q a in oxygen affinity and in autooxidation rate in sperm whale This to a for other amino acid that for the between the properties of the single H64Q sperm whale mutant and native elephant 1H NMR studies of elephant myoglobin the of in the distal of the heme which is not in other mammalian myoglobins. The with that with the et al., 1990; Vyas et al., The in of the distal and its was proposed to be the of the low autooxidation rate of elephant NMR of that it is at the position et al., The with this is that the published of elephant Mb Leu at this as is the in all other mammalian myoglobin et al., 1H NMR of L29F/H64Q is with that of elephant This correspondence between elephant myoglobin and the L29F/H64Q sperm whale mutant has been The for Phe29(B10) in L29F/H64Q are with those reported for in elephant Vyas et al. In the for elephant are from those of L29F/H64Q and the for both Gln64(E7) and are at position in elephant The for the elephant protein are also with those determined in the crystal structure of the L29F/H64Q mutant of sperm whale the of Phe29(B10) in elephant myoglobin has been by Bisig et have determined the structure of Asian elephant Thus, is a a is at position 29 (B10) in Asian elephant of the of chemical mechanisms in O2 and binding to myoglobin are in the binding of ligand of the distal pocket to and of the to form a The for the binding of both In the of this is by of a between of and the oxygen a O2 is in myoglobin than in model heme The between and is to the nature of the and for the of Thus, it is the of the distal and not as which in of O2 and binding et al., for O2 and The was from Springer et al. effects of mutagenesis are in of this In Gln64(E7) sperm whale myoglobin, the of the is from and in its position than the in the of the native protein et al., a O2 affinity is to The large in Phe29(B10) sperm whale myoglobin in the structure and the ligand et al., of these effects a of O2 In both the L29F/H64Q double mutant and elephant the to the of is by the of the A for the of elephant Mb to the double mutant is that by in elephant Mb is the of is to the distal of ligand than is the in the sperm whale double mutant et al., 1995). is to that binding is in the double mutant and elephant This is a of in affinity, rather than a in O2 affinity, compared to sperm whale The in is to of distal pocket in the form of the protein as a of by the residue and to with of autooxidation is shown in et al., the for autooxidation is of the and and the neutral In native the between the neutral of and the ligand the rate of autooxidation by of the since the for the is This same also O2 affinity by the same for the between and et al., Thus, to oxygen as a of the H64Q increases the rate of the increased the ligand and of to the replacement this the two either by mutagenesis or by in the of elephant myoglobin results in in compared to that of native sperm whale myoglobin for autooxidation. The was from et of in of in the distal pocket are also a of the for the in myoglobin et al., 1991; et al., 1994; Li et al., 1994; and Ling et al., in the order of the and a in the order of the as are the of the oxygen and Li et The in to the in the single Phe29(B10) mutant a in and in of the to the H64Q substitution in at and a of to both results in in the ligand compared to that in the protein as by the for and the of the and resonance Raman of elephant myoglobin and the L29F/H64Q mutant are distinct from those of native sperm whale myoglobin of the 1H NMR for the of the heme in all and the two native a molecular found by both x-ray crystallography and solution 1H NMR, the of in the distal pocket and structural in the of the distal et al., 1992; Quillin et al., 1993; et al., 1993; Qin et al., The NMR for L29F/H64Q a of the on the order of and in the of the as was for the mutant et al., The of the distal the The distal residues and between of the residues and the heme are the same as those for sperm whale myoglobin and Phe29(B10) in L29F/H64Q similar to those of and in the The of Gln to be for the of the a of the is in the crystal structure of the single mutant et al., The of the Gln64(E7) is both the solution and crystal structures of single and double In the is in a position to a to either in the or O2 in the of Phe29(B10) has a similar in the single and double of sperm whale myoglobin and in native elephant In all is to the to be in with the The in the NMR solution structure of the double mutant to be compared with that in the single mutant in This structural is by the of the Phe29(B10) to in L29F/H64Q than that in This in between the double and single is not in the crystal structure 1H NMR are to this between the solution and crystal and the of L29F/H64Q mutant was originally constructed to be a blood substitute prototype with moderately low O2 affinity, large rate constants, and increased resistance to autooxidation The on the basis of the mechanisms shown in The large Phe was at the 29 position to from the and the resistance to autooxidation. this single substitution is not it markedly increases oxygen affinity and myoglobin to et al., The H64Q was to in the of the Phe29(B10) of this was that native elephant myoglobin contains the same double In it that the elephant protein a since both of the single are to either high oxygen affinity or to oxidation also is and to that the same distal pocket combination which was rationally designed on the basis of proposed mechanisms for ligand binding and autooxidation is also in nature. INTRODUCTIONMyoglobin is a small, 16-18-kDa, globular hemeprotein which stores molecular oxygen in muscle cells. Because of the similarity of their tertiary structures, myoglobin is frequently used as a model for the α and β subunits of tetrameric hemoglobin. The key physiological properties of myoglobin are its high affinity for oxygen and low rate of spontaneous oxidation to the inactive ferric form (Antonini and Brunori, 1971). Studies of naturally occurring and mutant hemeproteins have shown that ligand affinity and autooxidation are affected markedly by the chemical nature of the amino acids in the heme pocket. In order to understand these functional properties, the molecular structures of a large number of myoglobins and hemoglobins have been determined by x-ray crystallography and solution NMR studies. These studies have been pursued along two lines. Traditionally, naturally occurring genetic or species variants with distinctive functional properties have been investigated (i.e. Bolognesi et al.(1989), Steigmann and Weber(1979), Vainshtein et al.(1978), Yu et al.(1990), Qin and La Mar (1992), and Vyas et al.(1993)). More recently, site-directed mutagenesis has been used either to test structural hypotheses (Olson et al., 1988; Braunstein et al., 1988; Carver et al., 1990; Ikeda-Saito et al., 1991; Balasubramanian et al., 1993; Li et al., 1994; Ling et al., 1994; Huang and Boxer, 1994) or to explore systematically the physiological roles of individual residues (Rohlfs et al., 1990; Egeberg et al., 1990; Smerdon et al., 1991, 1993; Carver et al., 1991, 1992; Quillin et al., 1993, 1995; Lai et al., 1995). A review of the mutagenesis literature is given in Springer et al.(1994).Dene et al.(1980) sequenced Asian elephant myoglobin and reported that it contains a Gln at position 64 (E7) rather than a His, which is found at this position in all other mammalian myoglobins. This replacement appeared to be neutral since the O2 affinity and autooxidation rate of elephant myoglobin are very similar to those of pig, human, and, sperm whale Mb 1The abbreviations used are: MbdeoxymyoglobinMbCOcarbonmonoxy et al., Springer et al., et that in elephant myoglobin, of Gln64(E7) a with oxygen which is in to that with in all other mammalian myoglobins. this was by the Gln in sperm whale myoglobin, the results that this substitution is not The H64Q a in in a Studies at H64Q sperm whale myoglobin not the functional and properties of elephant myoglobin have been pursued by two La Mar and et al., 1990; Vyas et al., have high 1H NMR studies of both and of elephant Mb and compared the results to native sperm whale myoglobin as a The in to for the distal are also which that a is in with ligand in the elephant and et al., 1992; et al., 1992; Springer et al., 1994; Li et al., 1994) have a of at position 29 (B10) in sperm whale of Phe for in sperm whale myoglobin to a in O2 affinity and in autooxidation rate et al., The crystal structure of this mutant that of the with The is by with the of the for the in affinity shown by the mutant. The large also from the distal pocket of the form of the mutant. structure of has been determined to by Quillin as a of The of the and Phe29(B10) are very similar to those reported for the and structures of this mutant. A of the structure is the of distal pocket to the large of the In native and sperm whale a is found to of and to the heme et al., The and effects also of the and et al., to a blood substitute and et al., 1993; 1994) constructed a double mutant in which Phe was at the 29 position to autooxidation by from the and Gln was at the 64 position to O2 affinity by to the it was that this was to in elephant Mb by Yu et the published for the elephant protein a Leu at the position et al., this and test the of Yu et al.(1990), we have compared in detail the spectral, and functional properties of elephant myoglobin and the L29F/H64Q sperm whale mutant. for O2 and binding and autooxidation determined by conventional kinetic of the of and L29F/H64Q sperm whale myoglobins compared with that of elephant Raman used to the and for the same of and native The structure of the of the form of the sperm whale double mutant was determined by solution 1H NMR and compared with that of the single and elephant the structure of L29F/H64Q sperm whale was determined by x-ray crystallography for with the determined by NMR and with the crystal structure of native elephant by Bisig et