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Histidine ligand variants of a flavo-diiron protein: effects on structure and activities.

ABSTRACT: Flavo-diiron proteins (FDPs) contain non-heme diiron and proximal flavin mononucleotide (FMN) active sites and function as terminal components of a nitric oxide reductase (NOR) and/or a four-electron dioxygen reductase (O(2)R). While most FDPs show similar structural, spectroscopic, and redox properties, O(2)R and NOR activities vary significantly among FDPs. A potential source of this variability is the iron ligation status of a conserved His residue that provides an iron ligand in all known FDP structures but one, where this His residue is rotated away from iron and replaced by a solvent ligand. In order to test the effect of this His ligation status, we changed this ligating His residue (H90) in Thermotoga maritima (Tm) FDP to either Asn or Ala. The wild-type Tm FDP shows significantly higher O(2)R than NOR activity. Single crystal X-ray crystallography revealed a remarkably conserved diiron site structure in the H90N and -A variants, differing mainly by either Asn or solvent coordination, respectively, in place of H90. The steady-state activities were minimally affected by the H90 substitutions, remaining significantly higher for O(2)R versus NOR. The pre-steady-state kinetics of the fully reduced FDP with O(2) were also minimally affected by the H90 substitutions. The results indicate that the coordination status of this His ligand does not significantly modulate the O(2)R or NOR activities, and that FDPs can retain these activities when the individual iron centers are differentiated by His ligand substitution. This differentiation may have implications for the O(2)R and NOR mechanisms of FDPs.

SUBMITTER: Fang H

PROVIDER: S-EPMC3508297 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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Histidine ligand variants of a flavo-diiron protein: effects on structure and activities.

Fang Han H Caranto Jonathan D JD Mendoza Rosalinda R Taylor Alexander B AB Hart P John PJ Kurtz Donald M DM

Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry 20120919 8

Flavo-diiron proteins (FDPs) contain non-heme diiron and proximal flavin mononucleotide (FMN) active sites and function as terminal components of a nitric oxide reductase (NOR) and/or a four-electron dioxygen reductase (O(2)R). While most FDPs show similar structural, spectroscopic, and redox properties, O(2)R and NOR activities vary significantly among FDPs. A potential source of this variability is the iron ligation status of a conserved His residue that provides an iron ligand in all known FD ...[more]

PMID: 22990880

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Project description:The unique active site of flavo-diiron proteins (FDPs) consists of a nonheme diiron-carboxylate site proximal to a flavin mononucleotide (FMN) cofactor. FDPs serve as the terminal components for reductive scavenging of dioxygen or nitric oxide to combat oxidative or nitrosative stress in bacteria, archaea, and some protozoan parasites. Nitric oxide is reduced to nitrous oxide by the four-electron reduced (FMNH2-Fe(II)Fe(II)) active site. In order to clarify the nitric oxide reductase mechanism, we undertook a multispectroscopic presteady-state investigation, including the first Mössbauer spectroscopic characterization of diiron redox intermediates in FDPs. A new transient intermediate was detected and determined to be an antiferromagnetically coupled diferrous-dinitrosyl (S = 0, [{FeNO}(7)]2) species. This species has an exchange energy, J ≥ 40 cm(-1) (JS1 ° S2), which is consistent with a hydroxo or oxo bridge between the two irons. The results show that the nitric oxide reductase reaction proceeds through successive formation of diferrous-mononitrosyl (S = ½, Fe(II){FeNO}(7)) and the S = 0 diferrous-dinitrosyl species. In the rate-determining process, the diferrous-dinitrosyl converts to diferric (Fe(III)Fe(III)) and by inference N2O. The proximal FMNH2 then rapidly rereduces the diferric site to diferrous (Fe(II)Fe(II)), which can undergo a second 2NO → N2O turnover. This pathway is consistent with previous results on the same deflavinated and flavinated FDP, which detected N2O as a product (Hayashi Biochemistry 2010, 49, 7040). Our results do not support other proposed mechanisms, which proceed either via "super-reduction" of [{FeNO}(7)]2 by FMNH2 or through Fe(II){FeNO}(7) directly to a diferric-hyponitrite intermediate. The results indicate that an S = 0 [{FeNO}(7)}]2 complex is a proximal precursor to N-N bond formation and N-O bond cleavage to give N2O and that this conversion can occur without redox participation of the FMN cofactor.

Project description:Two mechanisms, induced fit (IF) and conformational selection (CS), have been proposed to explain ligand recognition coupled conformational changes. The histidine binding protein (HisJ) adopts the CS mechanism, in which a pre-equilibrium is established between the open and the closed states with the ligand binding to the closed state. Despite being structurally similar to HisJ, the maltose binding protein (MBP) adopts the IF mechanism, in which the ligand binds the open state and induces a transition to the closed state. To understand the molecular determinants of this difference, we performed molecular dynamics (MD) simulations of coarse-grained dual structure based models. We find that intra-protein contacts unique to the closed state are sufficient to promote the conformational transition in HisJ, indicating a CS-like mechanism. In contrast, additional ligand-mimicking contacts are required to "induce" the conformational transition in MBP suggesting an IF-like mechanism. In agreement with experiments, destabilizing modifications to two structural features, the spine helix (SH) and the balancing interface (BI), present in MBP but absent in HisJ, reduce the need for ligand-mimicking contacts indicating that SH and BI act as structural restraints that keep MBP in the open state. We introduce an SH like element into HisJ and observe that this can impede the conformational transition increasing the importance of ligand-mimicking contacts. Similarly, simultaneous mutations to BI and SH in MBP reduce the barrier to conformational transitions significantly and promote a CS-like mechanism. Together, our results show that structural restraints present in the protein structure can determine the mechanism of conformational transitions and even simple models that correctly capture such structural features can predict their positions. MD simulations of such models can thus be used, in conjunction with mutational experiments, to regulate protein ligand interactions, and modulate ligand binding affinities.

Dataset Information

Histidine ligand variants of a flavo-diiron protein: effects on structure and activities.

Publications

Histidine ligand variants of a flavo-diiron protein: effects on structure and activities.

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