Amino acid replacement is a useful strategy to assess the roles of axial heme ligands in the function of native heme proteins. THB1, the protein product of the Chlamydomonas reinhardtii THB1 gene, is a group 1 truncated hemoglobin that uses a lysine residue in the E helix (Lys53, at position E10 by reference to myoglobin) as an iron ligand at neutral pH. Phylogenetic evidence shows that many homologous proteins have a histidine, methionine or arginine at the same position. In THB1, these amino acids would each be expected to convey distinct reactive properties if replacing the native lysine as an axial ligand. To explore the ability of the group 1 truncated Hb fold to support alternative ligation schemes and distal pocket conformations, the properties of the THB1 variants K53A as a control, K53H, K53M, and K53R were investigated by electronic absorption, EPR, and NMR spectroscopies. We found that His53 is capable of heme ligation in both the Fe(III) and Fe(II) states, that Met53 can coordinate only in the Fe(II) state, and that Arg53 stabilizes a hydroxide ligand in the Fe(III) state. The data illustrate that the group 1 truncated Hb fold can tolerate diverse rearrangement of the heme environment and has a strong tendency to use two protein side chains as iron ligands despite accompanying structural perturbations. Access to various redox pairs and different responses to pH make this protein an excellent test case for energetic and dynamic studies of heme ligation.

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 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.

The hemoglobin of Synechococcus sp. PCC 7002, GlbN, is a monomeric group I truncated protein (TrHb1) that coordinates the heme iron with two histidine ligands at neutral pH. One of these is the distal histidine (His46), a residue that can be displaced by dioxygen and other small molecules. Here, we show with mutagenesis, electronic absorption spectroscopy, and nuclear magnetic resonance (NMR) spectroscopy that at high pH and exclusively in the ferrous state, Lys42 competes with His46 for the iron coordination site. When b heme is originally present, the population of the lysine-bound species remains too small for detailed characterization; however, the population can be increased significantly by using dimethyl-esterified heme. Electronic absorption and NMR spectroscopies showed that the reversible ligand switching process occurs with an apparent pKa of 9.3 and a Lys-ligated population of ∼60% at the basic pH limit in the modified holoprotein. The switching rate, which is slow on the chemical shift time scale, was estimated to be 20-30 s-1 by NMR exchange spectroscopy. Lys42-His46 competition and attendant conformational rearrangement appeared to be related to weakened bis-histidine ligation and enhanced backbone dynamics in the ferrous protein. The pH- and redox-dependent ligand exchange process observed in GlbN illustrates the structural plasticity allowed by the TrHb1 fold and demonstrates the importance of electrostatic interactions at the heme periphery for achieving axial ligand selection. An analogy is drawn to the alkaline transition of cytochrome c, in which Lys-Met competition is detected at alkaline pH, but, in contrast to GlbN, in the ferric state only.

The cyanobacterium Synechococcus sp. PCC 7002 uses a monomeric hemoglobin, GlbN, to defend itself against reactive oxygen and nitrogen species. Bis-histidine coordination of the heme iron contributes to the resistance of the protein to oxidative damage. To characterize the stability of the iron ligation, we pursued pH titrations of GlbN and found by NMR spectroscopy that a lysine is able to displace the distal histidine from the ferrous iron, but not the ferric iron, at pH above 10.5. With mutagenesis, this lysine was identified as Lys42, a residue that interacts with one of the heme propionates in the bis-histidine complex. Indeed, when the heme is replaced with its dimethylester derivative, lysine ligation occurs at lower pH, which facilitated the study of the His-Fe-Lys complex. Absorbance spectroscopy was used to determine the apparent pKa of the conformational transition (9.2 ± 0.1), and NMR spectroscopy quantified the partitioning between the His-Fe-Lys and His-Fe-His states as well as the rate of interconversion. Slow exchange on the chemical shift time scale allowed for the description of key structural features. Ligand switching in GlbN is reminiscent of the alkaline transition of cytochrome c, in which one or more lysine residues displace the axial methionine in the ferric, but not ferrous, oxidation state. Selection of endogenous heme ligands has profound effects on the redox potential, electron transfer kinetics, and ligand binding reactions of a heme protein. Our results implicate the lysine-heme propionate interaction as an important determinant of the ligation state in GlbN.

Backgrount: The nuclear genome of Chlamydomonas reinhardtii encodes a dozen hemoglobins of the truncated lineage. Four of these, named THB1-4, contain a single similar to 130-residue globin unit. THB1, which is cytoplasmic and capable of nitric oxide dioxygenation activity, uses a histidine and a lysine as axial ligands to the heme iron. In the present report, we compared THB2, THB3, and THB4 to THB1 to gain structural and functional insights into algal globins. Methods: We inspected properties of the globin domains prepared by recombinant means through site-directed mutagenesis, electronic absorption, CD, and NMR spectroscopies, and X-ray crystallography. Results: Recombinant THB3, which lacks the proximal histidine but has a distal histidine, binds heme weakly. NMR data demonstrate that the recombinant domains of THB2 and THB4 coordinate the ferrous heme iron with the proximal histidine and a lysine from the distal helix. An X-ray structure of ferric THB4 confirms lysine coordination. THB1, THB2, and THB4 have reduction potentials between -65 and -100 mV, are capable of nitric oxide dioxygenation, are reduced at different rates by the diaphorase domain of C. reinhardtii nitrate reductase, and show different response to peroxide treatment. Conclusions: Three single-domain C. reinhardtii hemoglobins use lysine as a distal heme ligand in both Fe(III) and Fe(II) oxidation states. This common feature is likely related to enzymatic activity in the management of reactive oxygen species. General significance: Primary structure analysis of hemoglobins has limited power in the prediction of heme ligation. Experimental determination reveals variations in this essential property across the superfamily.

The cyanobacterium Synechococcus sp. PCC 7002 produces a monomeric hemoglobin (GlbN) implicated in the detoxification of reactive nitrogen and oxygen species. GlbN contains a b heme, which can be modified under certain reducing conditions. The modified protein (GlbN-A) has one heme–histidine C–N linkage similar to the C–S linkage of cytochrome c. No clear functional role has been assigned to this modification. Here, optical absorbance and NMR spectroscopies were used to compare the reactivity of GlbN and GlbN-A toward nitric oxide (NO). Both forms of the protein are capable of NO dioxygenase activity and both undergo heme bleaching after multiple NO challenges. GlbN and GlbN-A bind NO in the ferric state and form diamagnetic complexes (FeIII–NO) that resist reductive nitrosylation to the paramagnetic FeII–NO forms. Dithionite reduction of FeIII–NO GlbN and GlbN-A, however, resulted in distinct outcomes. Whereas GlbN-A rapidly formed the expected FeII–NO complex, NO binding to FeII GlbN caused immediate heme loss and, remarkably, was followed by slow heme rebinding and HNO (nitrosyl hydride) production. Additionally, combining FeIII GlbN, 15N-labeled nitrite, and excess dithionite resulted in the formation of FeII–H15NO GlbN. Dithionite-mediated HNO production was also observed for the related GlbN from Synechocystis sp. PCC 6803. Although ferrous GlbN-A appeared capable of trapping preformed HNO, the histidine–heme post-translational modification extinguished the NO reduction chemistry associated with GlbN. Overall, the results suggest a role for the covalent modification in FeII GlbNs: protection from NO-mediated heme loss and prevention of HNO formation.

GlbN, the "truncated" hemoglobin from Synechocystis sp. PCC 6803, coordinates the heme iron with His46 (E10, distal) and His70 (F8, proximal). Displacement of His E10 by an exogenous ligand (e.g., cyanide in the ferric state) drives a significant conformational change allowing Tyr22 (B10), Gln43 (E7) and Gln47 (E11) to establish a hydrogen bond network stabilizing the distal ligand. THB1, a closely related hemoglobin from Chlamydomonas reinhardtii, also binds cyanide and forms the same network of interactions. However, in the absence of an exogenous ligand, the neutral amino group of Lys53 (E10) coordinates the heme iron on the distal side [1]. Because of the importance of the coordination scheme in controlling the reactivity of heme proteins, the flexibility of the truncated hemoglobin fold, and the rarity of lysine coordination, we explored the ability of GlbN to use lysine at position E10 as an axial ligand. The His46Lys replacement yielded a protein with UV-Vis and NMR spectra similar to those of His46Leu and comparable pH response, suggesting that GlbN does not accommodate lysine coordination at position E10. However, the spectra were incompatible with water coordination and suggested that in the ferric state these variants were low-spin endogenously hexacoordinate complexes. We performed amino acid replacements within the distal H-bond network to characterize the perturbed heme environment and ligand sets. A combination of pH titrations and NMR experiments illustrates the delicate balance of interactions governing the heme pocket conformation of GlbN. [1] Johnson et al. (2014) Biochemistry 53:4573 Supported by NSF grant MCB-1330488

ABSTRACT Streptococcus mutans is the causative agent of dental caries, a significant concern for human health, and therefore an attractive target for therapeutics development. Previous work in our laboratory has identified a homodimeric, manganese-dependent repressor protein, SloR, as an important regulator of cariogenesis and has used site-directed mutagenesis to map functions to specific regions of the protein. Here we extend those studies to better understand the structural interaction between SloR and its operator and its effector metal ions. The results of DNase I assays indicate that SloR protects a 42-bp region of DNA that overlaps the sloABC promoter on the S. mutans UA159 chromosome, while electrophoretic mobility shift and solution binding assays indicate that each of two SloR dimers binds to this region. Real-time semiquantitative reverse transcriptase PCR (real-time semi-qRT-PCR) experiments were used to determine the individual base pairs that contribute to SloR-DNA binding specificity. Solution studies indicate that Mn 2+ is better than Zn 2+ at specifically activating SloR to bind DNA, and yet the 2.8-Å resolved crystal structure of SloR bound to Zn 2+ provides insight into the means by which selective activation by Mn 2+ may be achieved and into how SloR may form specific interactions with its operator. Taken together, these experimental observations are significant because they can inform rational drug design aimed at alleviating and/or preventing S. mutans -induced caries formation. IMPORTANCE This report focuses on investigating the SloR protein as a regulator of essential metal ion transport and virulence gene expression in the oral pathogen Streptococcus mutans and on revealing the details of SloR binding to its metal ion effectors and binding to DNA that together facilitate this expression. We used molecular and biochemical approaches to characterize the interaction of SloR with Mn 2+ and with its SloR recognition element to gain a clearer picture of the regulatory networks that optimize SloR-mediated metal ion homeostasis and virulence gene expression in S. mutans . These experiments can have a significant impact on caries treatment and/or prevention by revealing the S. mutans SloR-DNA binding interface as an appropriate target for the development of novel therapeutic interventions.

The nuclear genome of the model organism Chlamydomonas reinhardtii contains genes for a dozen hemoglobins of the truncated lineage. Of those, THB1 is known to be expressed, but the product and its function have not yet been characterized. We present mutagenesis, optical, and nuclear magnetic resonance data for the recombinant protein and show that at pH near neutral in the absence of added ligand, THB1 coordinates the heme iron with the canonical proximal histidine and a distal lysine. In the cyanomet state, THB1 is structurally similar to other known truncated hemoglobins, particularly the heme domain of Chlamydomonas eugametos LI637, a light-induced chloroplastic hemoglobin. Recombinant THB1 is capable of binding nitric oxide (NO(•)) in either the ferric or ferrous state and has efficient NO(•) dioxygenase activity. By using different C. reinhardtii strains and growth conditions, we demonstrate that the expression of THB1 is under the control of the NIT2 regulatory gene and that the hemoglobin is linked to the nitrogen assimilation pathway.