In this thesis the biophysical properties of four phytochromes were investigated to understand the enhancement mechanism of fluorescence in phytochrome-based near-infrared fluorescent proteins and to understand the contribution of the protonation heterogeneity to the function of phytochrome. Phytochromes are red/far-red photosensor proteins found in many organisms, such as plants, bacteria or fungi. The investigated phytochromes are Cph1 and Cph2 from cyanobacteria, as well as Agp1 and Agp2 from the Agrobacterium tumefaciens. The photosensor itself is a linear tetrapyrrole, which is bound to the photosensor module of phytochrome comprising the PAS, GAF and PHY domain (in short PGP) for Cph1, Agp1 and Agp2. In the first part (Chapter 4 and 5), we performed pH-titration of the linear tetrapyrrole chromophore in the Pr (red absorbing) state of different Cph1 PGP and Agp1 PGP constructs. Moreover, the role of the chromophore type such as biliverdin (BV) and phycocyanobilin (PCB) in phytochrome protonation heterogeneity in the Pr state was investigated using covalent and non-covalent PCB binding to Agp1 PGP V249C and wildtype (WT), respectively. The structural changes at various positions in Cph1 PGP and Agp1 PGP were measured as a function of pH using picosecond time resolved fluorescence anisotropy. We showed that only in Cph1 PGP, a direct correlation of chromophore deprotonation with pH-dependent conformational changes was found, but not in Agp1 PGP. In the second part (Chapter 6 and 7), we characterized the fluorescent properties of several variants from two different phytochrome families: phycocyanobilin-binding phytochromes (Cph1 and Cph2) and biliverdin-binding bacteriophytochromes (Agp1, und Agp2, as well as the variants of PAiRFP2), using steady-state and time-resolved fluorescence spectroscopy. The PCB adducts of Agp1 PGP variants were characterized to test the effect of chromophore type and conserved amino acids in the chromophore binding pocket on fluorescence quantum yield and fluorescence lifetime. Our results on Cph1 indicated that the combination of mutations known to enhance fluorescence in the cyanobacterial phytochrome Cph1 yield a quantum yield of about 17%. Also, our results confirmed a remarkable higher fluorescence quantum yield of phycocyanobilin-binding phytochromes (Cph1 and Cph2) compared to biliverdin-binding bacteriophytochromes (Agp2 and PAiRFP2), as it was shown in the previous studies.