Photocycle and Vectorial Proton Transfer in a Rhodopsin from the Eukaryote Oxyrrhis marina

Abstract

Retinylidene photoreceptors are ubiquitously present in marine protists as first documented by the identification of green proteorhodopsin (GPR). We present a detailed investigation of a rhodopsin from the protist Oxyrrhis marina (OR1) with respect to its spectroscopic properties and to its vectorial proton transport. Despite its homology to GPR, OR1's features differ markedly in its pH dependence. Protonation of the proton acceptor starts at pH below 4 and is sensitive to the ionic conditions. The mutation of a conserved histidine H62 did not influence the pK(a) value in a similar manner as in other proteorhodopsins where the charged histidine interacts with the proton acceptor forming the so-called His-Asp cluster. Mutational and pH-induced effects were further reflected in the temporal behavior upon light excitation ranging from femtoseconds to seconds. The primary photodynamics exhibits a high sensitivity to the environment of the proton acceptor D100 that are correlated to the different initial states. The mutation of the H62 does not affect photoisomerization at neutral pH. This is in agreement with NMR data indicating the absence of the His-Asp cluster. The subsequent steps in the photocycle revealed protonation reactions at the Schiff base coupled to proton pumping even at low pH. The main electrogenic steps are associated with the reprotonation of the Schiff base and internal proton donor. Hence, OR1 shows a different theme of the His-Asp organization where the low pK(a) of the proton acceptor is not dominated by this interaction, but by other electrostatic factors.

A simple protocol for the isolation of proteorhodopsins of the dinoflagellate Oxyrrhis marina

“…An absorbance maximum at 519 nm was registered for the recombinant wild-type protein. The shoulders toward shorter wavelengths in the spectra of B2, B3, and B4 in the current study were previously also reported by Jahnke et al [22]. The 280/519 nm ratios calculated for B2, B3, and B4 (if present) are rather high and most likely due to the presence of TX-100, which shows absorbance at 280 nm.…”

“…Oesterhelt and Stoeckenius [4] calculated a value of 2 for the 280/560 nm ratio of the bacteriorhodopsin of H. salinarum . Similar values of approximately 2 have been reported for the recombinant wild‐type protein and point‐mutated derivatives of ABV22426 by Janke et al [22]. These proteins, however, were heterologously expressed and purified by His‐tagged metal affinity chromatography.…”

“…This result is in line with the expected value and confirms that green light-absorbing proteorhodopsins were isolated. Janke et al [22] focused on the most abundant proteorhodopsin OR1 (equivalent to ABV22426) of the Oxyrrhis strain investigated. The authors cloned, expressed, and purified the wild-type protein and point-mutated derivatives thereof using the yeast Pichia pastoris and characterized them with respect to their spectroscopic properties and vectorial proton transport.…”

A new group of eubacterial light-driven retinal-binding proton pumps with an unusual cytoplasmic proton donor

“…More than a quarter of century later, a chain of discoveries established widespread presence of protonpumping rhodopsins in marine and freshwater eubacteria, and these proteins became known as proteorhodopsins (PR), xanthorhodopsins (XR) and actinorhodopsins [17][18][19][20]. In parallel, it was realized that various lower eukaryotes, including multiple fungi and some algae, possess retinalbinding light-driven proton pumps as well [21][22][23][24]. Biophysical studies of proton transport in these newly discovered rhodopsins provided information on the essential (conserved) and optional (variable) elements in its molecular mechanism [25][26][27].…”

The Evolution of Photosynthesis and Its Environmental Impact

“…Microbial rhodopsin was fi rst known from halorhodopsin in Halobacterium . A variant of this rhodopsin, known as proteorhodopsin, is found not only among α- (Stingl et al 2007 ), β-, and γ-proteobacteria, such as the classical organoheterotroph Vibrio campbellii (Wang et al 2012 ), but also among Bacteriodetes (Gómez-Consarnau et al 2007, González et al 2008Riedel et al 2010 ), Archaea , and eukaryotes (Janke et al 2013 ). Although proteorhodopsin is related to halorhodopsin and other "microbial" (type I) rhodopsins, it forms a very distinct clade (McCarren and DeLong 2007 ;Fig.…”