Seven mutant LH1 polypeptides of Rhodobactor sphaeroides have been isolated, and their behaviors in in vitro reconstitution of LH1 and its subunit complex have been characterized. Two mutants were selected to address the increased stability of the subunit complex of Rb. sphaeroides compared with that of Rhodobacter capsulatus. We found that this difference can be largely ascribed to the existence of Tyr at position +4 in the beta-polypeptide (the numbering system used assigns position 0 to the His which provides the coordinating ligand to bacteriochlorophyll) of the former bacterium compared to Met in that position in the latter. The amount of energy involved in the increased interaction was 1.6 kcal/mol, which would be consistent with a hydrogen bond involving Tyr. Mutation of the His at position 0 to Asn allows an estimate of the binding energy for subunit formation contributed by coordination of the imidazole group of His to the Mg atom of bacteriochlorophyll of >4.5 kcal/mol per BChl. Finally, an evaluation of the role of amino acids in the C-terminal region of the alpha-polypeptide was begun. Reconstitution of a mutant alpha-polypeptide in which Trp at position +11 was changed to Phe resulted in optimal formation of an LH1-type complex whose lambda(max) was blue-shifted to 853 nm, the same as observed in the intact bacterium harboring this mutation. These results provide further confirmation that the environment of BChl in reconstituted LH1 complexes is the same as in vivo and support the assignment of this residue to a role in hydrogen bonding with the C3(1) carbonyl group of BChl. Two other mutants of the alpha-polypeptide in which 5 and 14 amino acids in the C-terminus were deleted were also examined. These were of interest because the latter mutant, unlike the former, resulted in a low level of expression of LH1 in intact cells. However, with both of these mutant polypeptides, reconstitution appeared identical to that of the native system. In the case of the mutant shortened by 14 amino acids, a small blue-shift in lambda(max) to 861 nm was observed, again reproducing the blue-shift exhibited by the intact cells. Thus, these results suggest that the lowered levels of in vivo expression observed in these two mutants are due to reduced incorporation of the alpha-polypeptide into the membrane or its increased degradation, rather than to decreased stabilization of the LH1 complex.

In most purple bacteria, the core light-harvesting complex (LH1) differs from the peripheral light-harvesting complex (LH2) in spectral properties and amino acid sequences. In Rhodospirillum (Rs. )molischianum, however, the LH2 closely resembles the LH1 of many species in amino acid sequence identity and in some spectral properties (e.g., circular dichroism and resonance Raman). Despite these similarities to LH1, the LH2 of Rs. molischianum displays an absorption spectrum similar to the LH2 complexes of other bacteria. Moreover, its crystal structure is very similar to the LH2 of Rhodopseudomonas (Rps.) acidophila. To better understand the basis of the biochemical and spectral differences between LH1 and LH2, we isolated the alpha and beta polypeptides of the LH2 complexes from an LH2-only strain of Rhodobacter (Rb.) sphaeroides as well as the alpha and beta polypeptides from both the LH1 and LH2 complexes from Rs. molischianum. We then examined their behavior in reconstitution assays with bacteriochlorophyll (Bchl). The Rb. sphaeroides LH2 alpha and beta polypeptides were inactive in reconstitution assays, whether alone, paired with each other, or paired in hybrid assays with the complementary LH1 polypeptides of Rs. rubrum, Rb. sphaeroides, Rb. capsulatus, or Rps. viridis. The LH1 beta polypeptide of Rs. molischianum behaved similarly to the LH1 beta polypeptides of Rs. rubrum, Rb. sphaeroides, Rb. capsulatus, and Rps. viridis, forming a subunit-type complex with or without an alpha polypeptide, and forming an LH1 complex when combined with a native LH1 alpha polypeptide. Interestingly, the LH2 beta polypeptide of Rs. molischianum, in the absence of other polypeptides, also formed a subunit-type complex as well as a further red-shifted complex whose spectrum resembled the 850 nm absorbance band of LH2. In the presence of the LH1 alpha polypeptide of Rs. rubrum or Rs. molischianum, it formed an LH1-type complex, but in the presence of the LH2 alpha polypeptide of Rs. molischianum it formed an LH2 complex. This is the first reported reconstitution of an LH2 complex using only isolated LH2 polypeptides and Bchl. It is also the first example of an LH2 beta polypeptide that can form an LH1 subunit-type complex and an LH1-type complex when paired with an LH1 alpha polypeptide.

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