Ion Pair Formation between Basic Residues at 144 of the Cyt b Polypeptide and the Ubiquinones at the Q0 Site of the Cyt bc1 Complex
Loci of spontaneous Q0 site inhibitor resistant mutants in the cyt bc1 complex of the photosynthetic bacterium Rhodobacter capsulatus are M140, F144, G152, G158, and T163 of the cyt b polypeptide. In this report, we have studied the effects of arginine (R) substitution at these positions with a view to test for specific interactions with the [2Fe-2S] cluster, cyt bL with Q0 site ubiquinone (Q), or hydroquinone (QH2). All the arginine mutants displayed severely or completely impeded photosynthetic growth resulting from dysfunctional cyt bc1 complexes. The source of dysfunction in G158R and T163R was identified by a > 1000-fold decrease in the Q0 site affinity for QH2 and Q, sufficient to empty the site in the presence of the 30 mM ubiquinone pool of the chromatophore membrane; they appear similar to the class of mutants described in the preceding paper [Ding, H., Moser, C. C., Robertson, D. E., Tokito, M., Daldal, F., & Dutton, P. L (1995) Biochemistry 34, 15979-15996]. The source(s) of dysfunction of M140R and G152R is not so apparent since they possess Q0 sites with normal QH2/Q affinity; they appear to be members to the class of mutants identified and characterized in the following paper [Saribas, S., Ding, H., Dutton, P. L., & Daldal, F. (1995) Biochemistry 34, 16004-16012], The present paper focuses on the unique affects of F144R. Redox potential and EPR spectral properties of the Q0 site of F144R showed that arginine forms an ion pair with the head group of an anionic ubiquinone, tentatively suggested to be a ubihydroquinone anion (QH-) in the Qos domain. The redox potential of the putative QH-/Q couple appears to be raised by at least 220 mV above that observed in the wild-type Q0 site. The apparent p value of R(H+) in the R(H+)-QH- ion pair is 10.1. Lysine substituted at FI44 displayed parallel properties to that of F144R; the apparent pK value of K(H+) in the K(H+)-QH- ion pair is 9.2. Results indicate that the strong electrostatic interaction between R(H+) or K(H+) at position 144 and the QH- in the Q0 site is able to compensate for the opposing tendency of the neutral forms of R and K at position 144 to weaken the binding affinity of the Q0 site for Q or QH2 by > 1000-fold. In contrast, F144 substituted with the normally unprotonated histidine (F144H) neither forms an ion pair with the Q0 site occupant, at least down to neutral pH values nor measurably decreases the affinity. © 1995, American Chemical Society. All rights reserved.