FTIR characterization of the primary electron donor in double mutants combining the heterodimer HL(M202) with the LH(L131), HF(L168), FH(M197), or LH(M160) mutations

Abstract

Light-induced FTIR difference spectroscopy of the photooxidation of the primary electron donor P in Rhodobacter sphaeroides chromatophores has been performed to investigate the effect of possible addition or removal of hydrogen bonds to the C 9 and C 2 carbonyls of the bacteriochlorophyll (BChl)-bacteriopheophytin (BPhe) heterodimer in the His M202→Leu mutant. Four double mutants were studied combining the HL(M202) mutation with each of the following mutations: Leu L131→His which should add a hydrogen bond to the BChl molecule (P L) of the heterodimer, His L168→Phe which should remove an existing hydrogen bond to P L, Leu M160→His and Phe M197→His, each of which should add a hydrogen bond to the BPhe molecule of the heterodimer. None of the double mutants display the ≈2600 cm −1 electronic transition nor the IR marker bands at ≈1550, 1480, and 1295 cm −1 characteristic of the BChl dimeric state of P + in native reaction centers. The absence of these bands demonstrates that the double mutants indeed remain heterodimeric as in the single mutant HL(M202), i.e. the positive charge is localized on the BChl molecule of the heterodimer. For HL(M202)+HF(L168), changes are observed at around 1618 cm −1, supporting the conclusion that a hydrogen bond has been removed at the 2a-acetyl CO of P L. For all of the double mutants and in particular for HL(M202)+LH(L131), the 9-keto carbonyl mode of P L is found at 1695–1700 cm −1, thus implying that this group is not involved in a hydrogen bond in the ground state. Comparison of the IR carbonyl modes observed in the double mutant HL(M202)+LH(M160) relative to each of the single mutants HL(M202) and LH(M160) leads to a new interpretation for the split positive signal at 1722–1712 cm −1 in HL(M202). In this model, the 9-keto CO mode from P L + gives rise to the 1722 cm −1 peak while the 1712 cm −1 peak is tentatively assigned to the electrochromic shift of the keto carbonyl of the BPhe half of the HL(M202) heterodimer.