Molecular mechanisms underlying the green insect camouflage have puzzled researchers for over a century. Here, we isolated and identified a green water-soluble protein from the integument of bush-cricket Tettigonia cantans . De novo sequencing and cloning revealed a severely fragmented form of vitellogenins, ubiquitous and multifunctional, but still largely enigmatic glycolipoproteins essential for embryonic development and lacking structural characterization. The distinctive color of the identified chromoprotein results from binding of a remarkable combination of farnesylated bilins (recently identified, tentative heme A catabolites) and xanthophylls, which commensurably absorb light in the 600 to 700 nm and 400 to 550 nm spectral regions and thereby produce a hue that perfectly mimics foliage. The high-resolution crystal structure of this unique ~80 kDa dichromophoric protein, which we named “dibilinoxanthinin” (DBXN), revealed two DBXN protomers, each consisting of three polypeptides, with a novel fold enclosing a large hydrophobic cavity that accommodates two bilins, two luteins, and four phosphatidylcholines, all anchored by hydrogen bonds and giving DBXN unique biochemical and optical properties. Among the green insects tested, some contained yellow and blue chromophores in separate fractions, while others had green proteins similar to DBXN, although not necessarily of the same size. Surprisingly, we isolated and identified a larger vitellogenin proteoform with DBXN-like absorption, from the green huntsman spider Micrommata virescens . These data illustrate striking variations in the DBXN-related pigmentation mechanism among different green arthropods and suggest that vitellogenins may have undergone neofunctionalization, reflecting their potential for functional diversification.

The search of a putative physiological electron acceptor for thiocyanate dehydrogenase (TcDH) newly discovered in the thiocyanate-oxidizing bacteria Thioalkalivibrio paradoxus revealed an unusually large, single-heme cytochrome c (CytC552), which was co-purified with TcDH from the periplasm. Recombinant CytC552, produced in Escherichia coli as a mature protein without a signal peptide, has spectral properties similar to the endogenous protein and serves as an in vitro electron acceptor in the TcDH-catalyzed reaction. The CytC552 structure determined by NMR spectroscopy reveals significant differences compared to those of the typical class I bacterial cytochromes c: a high solvent accessible surface area for the heme group and so-called “intrinsically disordered” nature of the histidine-rich N- and C-terminal regions. Comparison of the signal splitting in the heteronuclear NMR spectra of oxidized, reduced, and TcDH-bound CytC552 reveals the heme axial methionine fluxionality. The TcDH binding site on the CytC552 surface was mapped using NMR chemical shift perturbations. Putative TcDH-CytC552 complexes were reconstructed by the information-driven docking approach and used for the analysis of effective electron transfer pathways. The best pathway includes the electron hopping through His528 and Tyr164 of TcDH, and His83 of CytC552 to the heme group in accordance with pH-dependence of TcDH activity with CytC552.

During determination of the thiocyanate dehydrogenase (TcDH) structure difficulties have occurred, related to the fact that enzyme crystals have been either twinned or strongly anisotropic. The diffraction quality of crystals can be improved by using mutant forms as objects of a study or by studying the structure of a related enzyme from another organism. Based on the analysis of the oligomeric structure of TcDH, the mutant forms of the enzyme that are promising for improving the diffraction properties have been proposed. The crystals have been obtained and the structures of the TcDH mutant forms with the substitutions T169A and K281A have been solved. The structure of the mutant form with the substitution T169A is found to be similar to the previously solved structures. In the structure of the mutant form with the substitution K281A, a change in the tetramer structure that made twinning impossible has been detected.