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

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.

X-ray Structure and Nuclear Magnetic Resonance Analysis of the Interaction Sites of the Ga-Substituted Cyanobacterial Ferredoxin.

In chloroplasts, ferredoxin (Fd) is reduced by Photosystem I (PSI) and oxidized by Fd-NADP(+) reductase (FNR) that is involved in NADP(+) reduction. To understand the structural basis for the dynamics and efficiency of the electron transfer reaction via Fd, we complementary used X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. In the NMR analysis of the formed electron transfer complex with Fd, the paramagnetic effect of the [2Fe-2S] cluster of Fd prevented us from detecting the NMR signals around the cluster. To solve this problem, the paramagnetic iron-sulfur cluster was replaced with a diamagnetic metal cluster. We determined the crystal structure of the Ga-substituted Fd (GaFd) from Synechocystis sp. PCC6803 at 1.62 Šresolution and verified its functional complementation using affinity chromatography. NMR analysis of the interaction sites on GaFd with PSI (molecular mass of ∼1 MDa) and FNR from Thermosynechococcus elongatus was achieved with high-field NMR spectroscopy. With reference to the interaction sites with FNR of Anabaena sp. PCC 7119 from the published crystal data, the interaction sites of Fd with FNR and PSI in solution can be classified into two types: (1) the core hydrophobic residues in the proximity of the metal center and (2) the hydrophilic residues surrounding the core. The former sites are shared in the Fd:FNR and Fd:PSI complex, while the latter ones are target-specific and not conserved on the residual level.

A sulfated cyanobacterial polysaccharide proven as a strong inhibitor of human complement activity in an in vitro assay.

Cyanobacterial exopolysaccharides are a rich source of, so far, widely unexplored polysaccharides. One of these exopolysaccharides is a highly sulfated, linear polysaccharide from Synechocystis aquatilis containing the amino sugar N-acetyl-fucosamine. Some sulfated polysaccharides and glycosaminoglycans are known to be inhibitors of the human complement system, which is an important part of the innate immune system. Defects in this system or misregulation can cause serious diseases. Therefore, new compounds with complement inhibiting activity and simple test assays are of great interest. Exopolysaccharides from S. aquatilis (arabinofucans) were compared to those from Synechocystis pevalekii (complex heteropolysaccharides) and the well-known complement inhibitor heparin. Investigations were performed with a modified ELISA test system based on a commercially available test kit quantifying the membrane attack complex. Hereby the testing becomes more stable, robust, reproducible, easier to handle and, for the first time, the effect of exopolysaccharides and heparin on the lectin pathway could be tested. The exopolysaccharides from S. aquatilis could be shown to be a 30 times stronger inhibitor of the classical pathway of the complement system compared to heparin (IC50 = 0.3 µg/mL vs. 9.2 µg/mL). These exopolysaccharides are also inhibitors of the lectin pathway (IC50 = 10.8 µg/mL) in which, however, heparin is more potent (IC50 = 2.0 µg/mL). Interestingly, these exopolysaccharides do not inhibit the alternative pathway. The exopolysaccharides from S. pevalekii are inactive in all pathways. Furthermore, partially hydrolyzed and desulfated exopolysaccharides from S. aquatilis were tested showing that a minimum molecular size and degree of sulfation are important for the inhibitory effects, whereas unspecific influences by complex formation of exopolysaccharides with calcium could be excluded.