Solution 1H NMR Study of the Influence of Distal Hydrogen Bonding and N Terminus Acetylation on the Active Site Electronic and Molecular Structure of Aplysia limacinaCyanomet Myoglobin

Bao D Nguyen,Zhicheng Xia,Francesca Cutruzzolá,Carlo Travaglini Allocatelli,Maurizio Brunori,Gerd N La Mar

doi:10.1074/jbc.275.2.742

Abstract

The sea hare Aplysia limacina possesses a myoglobin in which a distal H-bond is provided by Arg E10 rather than the common His E7. Solution (1)H NMR studies of the cyanomet complexes of true wild-type (WT), recombinant wild-type (rWT), and the V(E7)H/R(E10)T and V(E7)H mutants of Aplysia Mb designed to mimic the mammalian Mb heme pocket reveal that the distal His in the mutants is rotated out of the heme pocket and is unable to provide a stabilizing H-bond to bound ligand and that WT and rWT differ both in the thermodynamics of heme orientational disorder and in heme contact shift pattern. The mean of the four heme methyl shifts is shown to serve as a sensitive indicator of variations in distal H-bonding among a set of mutant cyanomet globins. The heme pocket perturbations in rWT relative to WT were traced to the absence of the N-terminal acetyl group in rWT that participates in an H-bond to the EF corner in WT. Analysis of dipolar contacts between heme and axial His and between heme and the protein matrix reveal a small approximately 2 degrees rotation of the axial His in rWT relative to true WT and a approximately 3 degrees rotation of the heme in the double mutant relative to rWT Mb. It is demonstrated that both the direction and magnitude of the rotation of the axial His relative to the heme can be determined from the change in the pattern of the contact-dominated heme methyl shift and from the dipolar-dominated heme meso-H shift. However, only NOE data can determine whether it is the His or heme that actually rotates in the protein matrix.

Highlights

Myoglobin (Mb)1 is a member of the globin family of proteins of approximately 140 –150 residues that encapsulate heme and exhibit a remarkably strongly conserved fold of seven to eight helices (A–H) despite a high variability in sequence [1,2,3]
The pattern of the dominant heme methyl contact shifts reflect the orientation of the axial His relative to the heme (i.e. ␾ in Fig. 1) (29 –33), and the pattern of the dominant meso-H dipolar shift reflects the orientation of the rhombic
We report on the 1H NMR spectra of the cyanomet complex of the Aplysia mutants V(E7)H-Mb and V(E7)H/ R(E10)T-Mb that show that the individual distal His E7 residues are oriented out of the heme pocket and cannot participate in any H-bonding interaction with the bound cyanide

Summary

Introduction

Myoglobin (Mb) is a member of the globin family of proteins of approximately 140 –150 residues that encapsulate heme and exhibit a remarkably strongly conserved fold of seven to eight helices (A–H) despite a high variability in sequence [1,2,3]. The Mbs from the sea hare Aplysia limacina [37], like those from Dolabela auricularia [38], possess a Val E7 but still exhibit high O2 affinity and reasonably slow O2 off-rates Both crystallography [12] and solution 1H NMR on Aplysia Mb [22, 39] and solution 1H NMR of Dolabela Mb [40] have demonstrated that Arg E10 can orient into the heme pocket and provide an H-bond to bound ligand. A question that naturally arises is whether the alternate H-bonding residues in sperm whale and Aplysia Mbs can be interchanged solely by interchanging the Val E7/Arg E10 in Aplysia with the His E7/Thr E10 of sperm whale Mb. Similar studies have shown that substitution of the key distal residues can in small or large part transfer an unusual functional property from one globin to another [17, 26, 28]. Engineering the second residue to convert the Aplysia Mb to mimic the sperm whale Mb pocket, V(E7)H/ R(E10)T-Mb, failed to either recover a significant fraction of the O2 affinity or retard the O2 off-rates relative to WT Aplysia Mb

Results

Discussion

Conclusion