Parameterization of a single H-bond in Orange Carotenoid Protein by atomic mutation reveals principles of evolutionary design of complex chemical photosystems

Introduction: Dissecting the intricate networks of covalent and non-covalent interactions that stabilize complex protein structures is notoriously difficult and requires subtle atomic-level exchanges to precisely affect local chemical functionality. The function of the Orange Carotenoid Protein (OCP), a light-driven photoswitch involved in cyanobacterial photoprotection, depends strongly on two H-bonds between the 4-ketolated xanthophyll cofactor and two highly conserved residues in the C-terminal domain (Trp288 and Tyr201).Method: By orthogonal translation, we replaced Trp288 in Synechocystis OCP with 3-benzothienyl-L-alanine (BTA), thereby exchanging the imino nitrogen for a sulphur atom.Results: Although the high-resolution (1.8 Å) crystal structure of the fully photoactive OCP-W288_BTA protein showed perfect isomorphism to the native structure, the spectroscopic and kinetic properties changed distinctly. We accurately parameterized the effects of the absence of a single H-bond on the spectroscopic and thermodynamic properties of OCP photoconversion and reveal general principles underlying the design of photoreceptors by natural evolution.Discussion: Such “molecular surgery” is superior over trial-and-error methods in hypothesis-driven research of complex chemical systems

Role of hydrogen bond alternation and charge transfer states in photoactivation of the Orange Carotenoid Protein

Here, we propose a possible photoactivation mechanism of a 35-kDa blue light-triggered photoreceptor, the Orange Carotenoid Protein (OCP), suggesting that the reaction involves the transient formation of a protonated ketocarotenoid (oxocarbenium cation) state. Taking advantage of engineering an OCP variant carrying the Y201W mutation, which shows superior spectroscopic and structural properties, it is shown that the presence of Trp201 augments the impact of one critical H-bond between the ketocarotenoid and the protein. This confers an unprecedented homogeneity of the dark-adapted OCP state and substantially increases the yield of the excited photoproduct S*, which is important for the productive photocycle to proceed. A 1.37 Å crystal structure of OCP Y201W combined with femtosecond time-resolved absorption spectroscopy, kinetic analysis, and deconvolution of the spectral intermediates, as well as extensive quantum chemical calculations incorporating the effect of the local electric field, highlighted the role of charge-transfer states during OCP photoconversion

Role of hydrogen bond alternation and charge transfer states in photoactivation of the Orange Carotenoid Protein

Here, we propose a possible photoactivation mechanism of a 35-kDa blue light-triggered photoreceptor, the Orange Carotenoid Protein (OCP), suggesting that the reaction involves the transient formation of a protonated ketocarotenoid (oxocarbenium cation) state. Taking advantage of engineering an OCP variant carrying the Y201W mutation, which shows superior spectroscopic and structural properties, it is shown that the presence of Trp201 augments the impact of one critical H-bond between the ketocarotenoid and the protein. This confers an unprecedented homogeneity of the dark-adapted OCP state and substantially increases the yield of the excited photoproduct S*, which is important for the productive photocycle to proceed. A 1.37 Å crystal structure of OCP Y201W combined with femtosecond time-resolved absorption spectroscopy, kinetic analysis, and deconvolution of the spectral intermediates, as well as extensive quantum chemical calculations incorporating the effect of the local electric field, highlighted the role of charge-transfer states during OCP photoconversion