Replacement of Putative Axial Ligands of Heme Iron in Yeast Cytochrome c1 by Site-Directed Mutagenesis1

120

135

To identify amino acid residues that influence the assembly or stability of the manganese cluster in photosystem II, we have generated site-directed mutations in the D1 polypeptide of the cyanobacterium, Synechocystis sp. PCC 6803. Indirect evidence has suggested that the D1 polypeptide provides some of the ligands that are required for metal binding. Mutations at position 170 of D1 were selected for characterization, since an aspartate to asparagine mutation (DN170D1) at this position completely abolishes photoautotrophic growth, while retention of a carboxylic acid at this position (aspartate to glutamate, DE170D1) supports photoautotrophic growth. Photosystem II particles were purified from control, DE170D1, and DN170D1 cells by a procedure that retains high rates of oxygen evolution activity in control particles [Noren, G.H., Boerner, R.J., & Barry, B.A. (1991) Biochemistry 30, 3943-3950]. Spectroscopic analysis shows that the tyrosine radical, Z+, which normally oxidizes the manganese cluster, is rapidly reduced in the DE170D1 mutant, but not in the DN170D1 mutant. A possible explanation of this block or dramatic decrease in the rate of electron transfer between the manganese cluster and tyrosine Z is an alteration in the properties of the metal center. Quantitation of manganese in these particles is consistent with aspartate 170 influencing the stability or assembly of the manganese cluster, since the aspartate to asparagine mutation results in a decrease in the manganese content per reaction center. Photosystem II particles from DN170D1 show a 60% decrease in the amount of specifically bound manganese per reaction center, when compared to control particles. Also, we observe a 70% decrease in the amount of specifically bound manganese per reaction center in partially purified DN170D1 particles and at least an 80% decrease in the amount of hydroxylamine-reducible manganese in DN170D1 thylakoid membranes. Single-turnover fluorescence assays and steady-state EPR measurements demonstrate that the remaining, endogenous manganese does not rapidly reduce tyrosine Z+ in the DN170D1 mutant. Additional evidence that aspartate 170 influences the assembly or stability of the metal site comes from analysis of the DE170D1 mutant. Although this mutant assembles a functional manganese cluster, as assessed by oxygen evolution and spectroscopic assays, the properties of the manganese site are perturbed.

Section: Discussionsupporting

confidence: 54%

Evidence from directed mutagenesis that aspartate 170 of the D1 polypeptide influences the assembly and/or stability of the manganese cluster in the photosynthetic water-splitting complex

Boerner¹,

Nguyen²,

Barry³

et al. 1992

120

135

“…There have been other studies related to the axial ligand replacement in c-type cytochromes using various approaches. Nakai et al (1990) used site-directed mutagenesis to replace Ml64 (corresponding to Ml83 in R. capsulatus) of cyt Ci from the yeast Saccharomyces cerevisiae with leucine and lysine and observed that these mutants were unable to grow on nonfermentable substrates. While they could detect the presence of the apoprotein in Western blots of total cell homogenates, they were unable to detect an a-band in redox difference spectra for M164L but M164K had a very small a-band.…”

Section: Discussionmentioning

confidence: 99%

Mutagenesis of methionine-183 drastically affects the physicochemical properties of cytochrome c1 of the bc1 complex of Rhodobacter capsulatus

Gray¹,

Davidson²,

Daldal³

1992

Site-directed mutagenesis was used to investigate which of the highly conserved methionine residues (M183 and M205) provides the sixth axial ligand to the heme Fe in the cyt c1 subunit of the bc1 complex from the bacterium Rhodobacter capsulatus. These residues were changed to leucine (cM183L) and valine (cM205V). Two additional mutants were constructed, 1 in which a stop codon was inserted at M205 (cM205*) and the second in which 127 amino acids were deleted between the signal sequence and the putative C-terminal transmembrane alpha-helix (c delta SfuI). Only cM205V grew photosynthetically, and membranes isolated from this strain catalyzed quinol-dependent reduction of cyt c in amounts similar to that in a wild-type strain. Even though cM183L could not grow photosynthetically, it contained all the appropriate polypeptides and cofactors of the bc1 complex, as shown by SDS-PAGE and optical difference spectroscopy of intact membrane particles. Neither of the two deletion mutants contained a stable complex. Flash absorption spectroscopy using chromatophores showed no cytochrome c rereduction after oxidation by the reaction center in cM183L. The bc1 complex from each strain was isolated and characterized. Oxidation reduction midpoint potential titrations revealed that cyt c1 from cM183L had a dramatically shifted Em value (delta Em = -390 mV) compared with wild type and cM205V. While the optical absorption spectrum of cyt c1 from cM183L suggested that the c-type heme was low-spin, nonetheless it was able to react with the exogenous ligand carbon monoxide. The overall data support that M183, and not M205, is the sixth ligand to the heme Fe of cyt c1 of the bc1 complex.

“…Assembly and Heme Content of Mutant bc 1 Complexes. In yeast, it is known that mutation of the sixth axial ligand methionine of cyt c 1 to leucine interferes with the covalent attachment of heme, and leads to the accumulation of its precursor form (19). Thus, it seemed important to determine whether the lack of the bc 1 complex activity in R. capsulatus M183H or M183K mutants was due to the absence of a properly assembled bc 1 complex.…”

Section: Choice and Construction Of The M183h And M183kmentioning

confidence: 99%

“…On the other hand, very little work has been carried out on membrane-anchored c-type cytochromes such as the cyt c 1 subunit of the bc 1 complex, where proper incorporation of the mature hemoprotein into a multisubunit complex may be elaborate. Previously, only a few mutants affecting cyt c 1 in Saccharomyces cereVisiae (19), R. capsulatus (11), and Rhodobacter sphaeroides (20) have been studied. In particular, substitution of M183 with leucine (L) in the case of R. capsulatus yielded a cyt c 1 mutant with a drastically shifted E m7 value (∆E m7 ) -390 mV), and an ability to bind CO, not observed with the wild-type protein.…”

mentioning

confidence: 99%

Substitution of the Sixth Axial Ligand of Rhodobacter capsulatus Cytochrome c₁ Heme Yields Novel Cytochrome c₁ Variants with Unusual Properties

Darrouzet

Mandaci

et al. 1999

The cytochrome (cyt) c1 heme of the ubihydroquinone:cytochrome c oxidoreductase (bc1 complex) is covalently attached to two cysteine residues of the cyt c1 polypeptide chain via two thioether bonds, and the fifth and sixth axial ligands of its iron atom are histidine (H) and methionine (M), respectively. The latter residue is M183 in Rhodobacter capsulatus cyt c1, and previous mutagenesis studies revealed its critical role for the physicochemical properties of cyt c1 [Gray, K. A., Davidson, E., and Daldal, F. (1992) Biochemistry 31, 11864-11873]. In the homologous chloroplast b6f complex, the sixth axial ligand is provided by the amino group of the amino terminal tyrosine residue. To further pursue our investigation on the role played by the sixth axial ligand in heme-protein interactions, novel cyt c1 variants with histidine-lysine (K) and histidine-histidine axial coordination were sought. Using a R. capsulatus genetic system, the cyt c1 mutants M183K and M183H were constructed by site-directed mutagenesis, and chromatophore membranes as well as purified bc1 complexes obtained from these mutants were characterized in detail. The studies revealed that these mutants incorporated the heme group into the mature cyt c1 polypeptides, but yielded nonfunctional bc1 complexes with unusual spectroscopic and thermodynamic properties, including shifted optical absorption maxima (lambdamax) and decreased redox midpoint potential values (Em7). The availability and future detailed studies of these stable cyt c1 mutants should contribute to our understanding of how different factors influence the physicochemical and folding properties of membrane-bound c-type cytochromes in general.