Thiocyanate hydrolase (SCNase) purified from Thiobacillus thioparus THI115 hydrolyzes thiocyanate to carbonyl sulfide and ammonia. DNA sequences of the cloned genes revealed the close relation of SCNase to nitrile hydratase (NHase). The consensus sequences for coordination of the metal ion found in NHases were also conserved in the gamma subunit of SCNase. Here, we showed that the SCNase contained one cobalt atom per alphabetagamma heterotrimer. UV-vis absorption spectrum suggested that the cobalt exists as a non-corrin ion. Reduced SCNase showed an ESR signal characteristic of low-spin Co2+, which closely resembled that of the Co-type NHases. Mass spectrometry for the peptide fragment containing the metal-binding motif of the SCNase gamma subunit indicated that the cysteine residue at position 131 was post-translationally oxidized to a cysteine-sulfinic acid. From these results, we concluded that SCNases and NHases form a novel non-corrin and/or non-heme protein family having post-translationally modified cysteine ligands.

The native core light-harvesting complex (LH1) from the thermophilic purple phototrophic bacterium requires Ca for its thermal stability and characteristic absorption maximum at 915 nm. To explore the role of specific amino acid residues of the LH1 polypeptides in Ca-binding behavior, we constructed a genetic system for heterologously expressing the LH1 complex in an engineered mutant strain. This system contained a chimeric gene cluster ( from and from ) and was subsequently deployed for introducing site-directed mutations on the LH1 polypeptides. All mutant strains were capable of phototrophic (anoxic/light) growth. The heterologously expressed wild-type LH1 complex was isolated in a reaction center (RC)-associated form and displayed the characteristic absorption properties of this thermophilic phototroph. Spheroidene (the major carotenoid in ) was incorporated into the LH1 complex in place of its native spirilloxanthins with one carotenoid molecule present per αβ-subunit. The hybrid LH1-RC complexes expressed in were characterized using absorption, fluorescence excitation, and resonance Raman spectroscopy. Site-specific mutagenesis combined with spectroscopic measurements revealed that α-D49, β-L46, and a deletion at position 43 of the α-polypeptide play critical roles in Ca binding in the LH1 complex; in contrast, α-N50 does not participate in Ca coordination. These findings build on recent structural data obtained from a high-resolution crystallographic structure of the membrane integrated LH1-RC complex and have unambiguously identified the location of Ca within this key antenna complex.

The light-harvesting 1 reaction center (LH1-RC) complex from Thermochromatium tepidum exhibits a largely red-shifted LH1 Q absorption at 915 nm due to binding of Ca, resulting in an "uphill" energy transfer from LH1 to the reaction center (RC). In a recent study, we developed a heterologous expression system (strain TS2) to construct a functional hybrid LH1-RC with LH1 from Tch. tepidum and the RC from Rhodobacter sphaeroides [Nagashima, K. V. P., et al. (2017) Proc. Natl. Acad. Sci. U. S. A. 114, 10906]. Here, we present detailed characterizations of the hybrid LH1-RC from strain TS2. Effects of metal cations on the phototrophic growth of strain TS2 revealed that Ca is an indispensable element for its growth, which is also true for Tch. tepidum but not for Rba. sphaeroides. The thermal stability of the TS2 LH1-RC was strongly dependent on Ca in a manner similar to that of the native Tch. tepidum, but interactions between the heterologous LH1 and RC became relatively weaker in strain TS2. A Fourier transform infrared analysis demonstrated that the Ca-binding site of TS2 LH1 was similar but not identical to that of Tch. tepidum. Steady-state and time-resolved fluorescence measurements revealed that the uphill energy transfer rate from LH1 to the RC was related to the energy gap in an order of Rba. sphaeroides, Tch. tepidum, and strain TS2; however, the quantum yields of LH1 fluorescence did not exhibit such a correlation. On the basis of these findings, we discuss the roles of Ca, interactions between LH1 and the RC from different species, and the uphill energy transfer mechanisms.