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Significantly enhanced heme retention ability of myoglobin engineered to mimic the third covalent linkage by nonaxial histidine to heme (vinyl) in synechocystis hemoglobin.

ABSTRACT: Heme proteins, which reversibly bind oxygen and display a particular fold originally identified in myoglobin (Mb), characterize the "hemoglobin (Hb) superfamily." The long known and widely investigated Hb superfamily, however, has been enriched by the discovery and investigation of new classes and members. Truncated Hbs typify such novel classes and exhibit a distinct two-on-two ?-helical fold. The truncated Hb from the freshwater cyanobacterium Synechocystis exhibits hexacoordinate heme chemistry and bears an unusual covalent bond between the nonaxial His(117) and a heme porphyrin 2-vinyl atom, which remains tightly associated with the globin unlike any other. It seems to be the most stable Hb known to date, and His(117) is the dominant force holding the heme. Mutations of amino acid residues in the vicinity did not influence this covalent linkage. Introduction of a nonaxial His into sperm whale Mb at the topologically equivalent position and in close proximity to vinyl group significantly increased the heme stability of this prototype globin. Reversed phase chromatography, electrospray ionization-MS, and MALDI-TOF analyses confirmed the presence of covalent linkage in Mb I107H. The Mb mutant with the engineered covalent linkage was stable to denaturants and exhibited ligand binding and auto-oxidation rates similar to the wild type protein. This indeed is a novel finding and provides a new perspective to the evolution of Hbs. The successful attempt at engineering heme stability holds promise for the production of stable Hb-based blood substitute.

SUBMITTER: Uppal S

PROVIDER: S-EPMC4303654 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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Significantly enhanced heme retention ability of myoglobin engineered to mimic the third covalent linkage by nonaxial histidine to heme (vinyl) in synechocystis hemoglobin.

Uppal Sheetal S Salhotra Shikha S Mukhi Nitika N Zaidi Fatima Kamal FK Seal Manas M Dey Somdatta Ghosh SG Bhat Rajiv R Kundu Suman S

The Journal of biological chemistry 20141201 4

Heme proteins, which reversibly bind oxygen and display a particular fold originally identified in myoglobin (Mb), characterize the "hemoglobin (Hb) superfamily." The long known and widely investigated Hb superfamily, however, has been enriched by the discovery and investigation of new classes and members. Truncated Hbs typify such novel classes and exhibit a distinct two-on-two α-helical fold. The truncated Hb from the freshwater cyanobacterium Synechocystis exhibits hexacoordinate heme chemist ...[more]

PMID: 25451928

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Project description:It is now well-established that mammalian heme proteins are reactive with various nitrogen oxide species and that these reactions may play significant roles in mammalian physiology. For example, the ferrous heme protein myoglobin (Mb) has been shown to reduce nitrite (NO(2)(-)) to nitric oxide (NO) under hypoxic conditions. We demonstrate here that the distal pocket histidine residue (His64) of horse heart metMb(III) (i.e., ferric Mb(III)) has marked effects on the mode of nitrite ion coordination to the iron center. X-ray crystal structures were determined for the mutant proteins metMb(III) H64V (2.0 A resolution) and its nitrite ion adduct metMb(III) H64V-nitrite (1.95 A resolution), and metMb(III) H64V/V67R (1.9 A resolution) and its nitrite ion adduct metMb(III) H64V/V67R-nitrite (2.0 A resolution). These are compared to the known structures of wild-type (wt) hh metMb(III) and its nitrite ion adduct hh metMb(III)-nitrite, which binds NO(2)(-) via an O-atom in a trans-FeONO configuration. Unlike wt metMb(III), no axial H(2)O is evident in either of the metMb(III) mutant structures. In the ferric H64V-nitrite structure, replacement of the distal His residue with Val alters the binding mode of nitrite from the nitrito (O-binding) form in the wild-type protein to a weakly bound nitro (N-binding) form. Reintroducing a H-bonding residue in the H64V/V67R double mutant restores the O-binding mode of nitrite. We have also examined the effects of these mutations on reactivities of the metMb(III)s with cysteine as a reducing agent and of the (ferrous) Mb(II)s with nitrite ion under anaerobic conditions. The Mb(II)s were generated by reduction of the Mb(III) precursors in a second-order reaction with cysteine, the rate constants for this step following the order H64V/V67R > H64V >> wt. The rate constants for the oxidation of the Mb(II)s by nitrite (giving NO as the other product) follow the order wt > H64V/V67R >> H64V and suggest a significant role of the distal pocket H-bonding residue in nitrite reduction.

Dataset Information

Significantly enhanced heme retention ability of myoglobin engineered to mimic the third covalent linkage by nonaxial histidine to heme (vinyl) in synechocystis hemoglobin.

Publications

Significantly enhanced heme retention ability of myoglobin engineered to mimic the third covalent linkage by nonaxial histidine to heme (vinyl) in synechocystis hemoglobin.

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