Investigation of protonatable residues in Rhodothermus marinus caa ₃ haem-copper oxygen reductase: Comparism with Paracoccus denitrificans aa₃ haem-copper oxygen reductase

The Rhodothermus marinus caa₃ haem-copper oxygen reductase contains all the residues of the so-called D- and K-proton channels, with the notable exception of the helix VI glutamate residue (Glu278^I in Paracoccus denitrificans aa₃), being nevertheless a true oxygen reductase reducing O₂ to water, and an efficient proton pump. Instead, in the same helix, but one turn below, it has a tyrosine residue (Tyr256¹, R. marinus caa₃ numbering), whose hydroxyl group occupies the same spatial position as the carboxylate group of Glu278 ^I, as deduced by comparative modelling techniques. Therefore, we proposed previously that this tyrosine residue could play an important role in the proton pathway. In this article we further study this hypothesis, by investigating the equilibrium thermodynamics of protonation in R. marinus caa₃, using theoretical methodologies based on the structural model previously obtained. Control calculations are also performed for the P. denitrificans aa3 oxygen reductase. In both oxygen reductases we find several residues that are proton active (i.e., that display partial protonation) at physiological pH, some of them being redox sensitive (i.e., sensitive to the protein redox state). However, the caa₃ Tyr256^I is not proton active at physiological pH, in contrast to the aa₃ Glu278 ^I which is both proton active at physiological pH and shows a high redox sensitivity. In R. marinus caa₃ we do not find any other residues in the same protein zone that can have this property. Therefore, there are no putative D-channel residues that are proton active in this oxidase. The protonatable residues of the K-channel are much more functionally conserved in both oxygen reductases than the same type of residues in the D-channel. Two (Tyr262^I and Lys336^I, caa₃ numbering) out of three protonatable K-channel residues are proton active and redox sensitive in both proteins.