Structural and kinetic properties of Rhodobacter sphaeroides photosynthetic reaction centers containing exclusively Zn-coordinated bacteriochlorophyll as bacteriochlorin cofactors.

The Zn-BChl-containing reaction center (RC) produced in a bchD (magnesium chelatase) mutant of Rhodobacter sphaeroides assembles with six Zn-bacteriochlorophylls (Zn-BChls) in place of four Mg-containing bacteriochlorophylls (BChls) and two bacteriopheophytins (BPhes). This protein presents unique opportunities for studying biological electron transfer, as Zn-containing chlorins can exist in 4-, 5-, and (theoretically) 6-coordinate states within the RC. In this paper, the electron transfer perturbations attributed exclusively to coordination state effects are separated from those attributed to the presence, absence, or type of metal in the bacteriochlorin at the HA pocket of the RC. The presence of a 4-coordinate Zn(2+) ion in the HA bacteriochlorin instead of BPhe results in a small decrease in the rates of the P*→P(+)HA(-)→P(+)QA(-) electron transfer, and the charge separation yield is not greatly perturbed; however coordination of the Zn(2+) by a fifth ligand provided by a histidine residue results in a larger rate decrease and yield loss. We also report the first crystal structure of a Zn-BChl-containing RC, confirming that the HA Zn-BChl was either 4- or 5-coordinate in the two types of Zn-BChl-containing RCs studied here. Interestingly, a large degree of disorder, in combination with a relatively weak anomalous difference electron density was found in the HB pocket. These data, in combination with spectroscopic results, indicate partial occupancy of this binding pocket. These findings provide insights into the use of BPhe as the bacteriochlorin pigment of choice at HA in both BChl- and Zn-BChl-containing RCs found in nature.

Role of Rhodobacter sphaeroides photosynthetic reaction center residue M214 in the composition, absorbance properties, and conformations of H(A) and B(A) cofactors.

In the native reaction center (RC) of Rhodobacter sphaeroides, the side chain of (M)L214 projects orthogonally toward the plane and into the center of the A branch bacteriopheophytin (BPhe) macrocycle. The possibility that this side chain is responsible for the dechelation of the central Mg(2+) of bacteriochlorophyll (BChl) was investigated by replacement of (M)214 with residues possessing small, nonpolar side chains that can neither coordinate nor block access to the central metal ion. The (M)L214 side chain was also replaced with Cys, Gln, and Asn to evaluate further the requirements for assembly of the RC with BChl in the HA pocket. Photoheterotrophic growth studies showed no difference in growth rates of the (M)214 nonpolar mutants at a low light intensity, but the growth of the amide-containing mutants was impaired. The absorbance spectra of purified RCs indicated that although absorbance changes are associated with the nonpolar mutations, the nonpolar mutant RC pigment compositions are the same as in the wild-type protein. Crystal structures of the (M)L214G, (M)L214A, and (M)L214N mutants were determined (determined to 2.2-2.85 Å resolution), confirming the presence of BPhe in the HA pocket and revealing alternative conformations of the phytyl tail of the accessory BChl in the BA site of these nonpolar mutants. Our results demonstrate that (i) BChl is converted to BPhe in a manner independent of the aliphatic side chain length of nonpolar residues replacing (M)214, (ii) BChl replaces BPhe if residue (M)214 has an amide-bearing side chain, (iii) (M)214 side chains containing sulfur are not sufficient to bind BChl in the HA pocket, and (iv) the (M)214 side chain influences the conformation of the phytyl tail of the BA BChl.

A BchD (magnesium chelatase) mutant of rhodobacter sphaeroides synthesizes zinc bacteriochlorophyll through novel zinc-containing intermediates.

Heme and bacteriochlorophyll a (BChl) biosyntheses share the same pathway to protoporphyrin IX, which then branches as follows. Fe(2+) chelation into the macrocycle by ferrochelatase results in heme formation, and Mg(2+) addition by Mg-chelatase commits the porphyrin to BChl synthesis. It was recently discovered that a bchD (Mg-chelatase) mutant of Rhodobacter sphaeroides produces an alternative BChl in which Mg(2+) is substituted by Zn(2+). Zn-BChl has been found in only one other organism before, the acidophilic Acidiphilium rubrum. Our objectives in this work on the bchD mutant were to 1) elucidate the Zn-BChl biosynthetic pathway in this organism and 2) understand causes for the low amounts of Zn-BChl produced. The bchD mutant was found to contain a Zn-protoporphyrin IX pool, analogous to the Mg-protoporphyrin IX pool found in the wild type strain. Inhibition of ferrochelatase with N-methylprotoporphyrin IX caused Zn-protoporphyrin IX and Zn-BChl levels to decline by 80-90% in the bchD mutant, whereas in the wild type strain, Mg-protoporphyrin IX and Mg-BChl levels increased by 170-240%. Two early metabolites of the Zn-BChl pathway were isolated from the bchD mutant and identified as Zn-protoporphyrin IX monomethyl ester and divinyl-Zn-protochlorophyllide. Our data support a model in which ferrochelatase synthesizes Zn-protoporphyrin IX, and this metabolite is acted on by enzymes of the BChl pathway to produce Zn-BChl. Finally, the low amounts of Zn-BChl in the bchD mutant may be due, at least in part, to a bottleneck upstream of the step where divinyl-Zn-protochlorophyllide is converted to monovinyl-Zn-protochlorophyllide.