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Replacement of the Distal Histidine Reveals a Noncanonical Heme Binding Site in a 2-on-2 Hemoglobin.

ABSTRACT: Heme ligation in hemoglobin is typically assumed by the "proximal" histidine. Hydrophobic contacts, ionic interactions, and the ligation bond secure the heme between two ?-helices denoted E and F. Across the hemoglobin superfamily, several proteins also use a "distal" histidine, making the native state a bis-histidine complex. The group 1 truncated hemoglobin from Synechocystis sp. PCC 6803, GlbN, is one such bis-histidine protein. Ferric GlbN, in which the distal histidine (His46 or E10) has been replaced with a leucine, though expected to bind a water molecule and yield a high-spin iron complex at neutral pH, has low-spin spectral properties. Here, we applied nuclear magnetic resonance and electronic absorption spectroscopic methods to GlbN modified with heme and amino acid replacements to identify the distal ligand in H46L GlbN. We found that His117, a residue located in the C-terminal portion of the protein and on the proximal side of the heme, is responsible for the formation of an alternative bis-histidine complex. Simultaneous coordination by His70 and His117 situates the heme in a binding site different from the canonical site. This new holoprotein form is achieved with only local conformational changes. Heme affinity in the alternative site is weaker than in the normal site, likely because of strained coordination and a reduced number of specific heme-protein interactions. The observation of an unconventional heme binding site has important implications for the interpretation of mutagenesis results and globin homology modeling.

SUBMITTER: Nye DB

PROVIDER: S-EPMC6217817 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Replacement of the Distal Histidine Reveals a Noncanonical Heme Binding Site in a 2-on-2 Hemoglobin.

Nye Dillon B DB Lecomte Juliette T J JTJ

Biochemistry 20180928 40

Heme ligation in hemoglobin is typically assumed by the "proximal" histidine. Hydrophobic contacts, ionic interactions, and the ligation bond secure the heme between two α-helices denoted E and F. Across the hemoglobin superfamily, several proteins also use a "distal" histidine, making the native state a bis-histidine complex. The group 1 truncated hemoglobin from Synechocystis sp. PCC 6803, GlbN, is one such bis-histidine protein. Ferric GlbN, in which the distal histidine (His46 or E10) has be ...[more]

PMID: 30213188

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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

Replacement of the Distal Histidine Reveals a Noncanonical Heme Binding Site in a 2-on-2 Hemoglobin.

Publications

Replacement of the Distal Histidine Reveals a Noncanonical Heme Binding Site in a 2-on-2 Hemoglobin.

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