Abstract
The influence of the amino acid residues sandwiching the flavin ring in flavodoxin (Fld) from the cyanobacterium Anabaena sp. PCC 7119 in complex formation and electron transfer (ET) with its natural partners, photosystem I (PSI) and ferredoxin-NADP(+) reductase (FNR), was examined in mutants of the key residues Trp(57) and Tyr(94). The mutants' ability to form complexes with either FNR or PSI is similar to that of wild-type Fld. However, some of the mutants exhibit altered kinetic properties in their ET processes that can be explained in terms of altered flavin accessibility and/or thermodynamic parameters. The most noticeable alteration is produced upon replacement of Tyr(94) by alanine. In this mutant, the processes that involve the transfer of one electron from either PSI or FNR are clearly accelerated, which might be attributable to a larger accessibility of the flavin to the reductant. However, when the opposite ET flow is analyzed with FNR, the reduced Y94A mutant transfers electrons to FNR slightly more slowly than wild type. This can be explained thermodynamically from a decrease in driving force due to the significant shift of 137 mV in the reduction potential value for the semiquinone/hydroquinone couple (E(1)) of Y94A, relative to wild type (Lostao, A., Gómez-Moreno, C., Mayhew, S. G., and Sancho, J. (1997) Biochemistry 36, 14334-14344). The behavior of the rest of the mutants can be explained in the same way. Overall, our data indicate that Trp(57) and Tyr(94) do not play any active role in flavodoxin redox reactions providing a path for the electrons but are rather involved in setting an appropriate structural and electronic environment that modulates in vivo ET from PSI to FNR while providing a tight FMN binding.
Highlights
The influence of the amino acid residues sandwiching the flavin ring in flavodoxin (Fld) from the cyanobacterium Anabaena sp
The Km values obtained for ferredoxin-NADP؉ reductase (FNR), using the different Fld variants as protein carriers in the NADPH-dependent cytochrome c reductase assay (Table I), as well as the data derived from differential spectroscopic analysis of the FNRox-Fldox interaction (Fig. 1, Table II) indicate that the introduced mutations do not produce any major effect in the stability of the FNRox1⁄7Fldox complex or in the functionality of the FNRrd1⁄7Fldox complex
In the case of the Y94A mutant, the introduced side chain does not provide an electronic environment that can improve by itself the electron transfer (ET) process, structural and thermodynamic aspects have to account for the enhancement observed
Summary
Dissociation constants, binding energies, and changes in extinction coefficients for the complexes between WT FNRox and the different Fldox variants were obtained by differential spectroscopy as previously described [18]. Steady-state Enzymatic Assays—The NADPH-dependent cytochrome c reductase activity of FNR was determined using the different Fld mutants as the electron carrier from FNR to cytochrome c [18, 23]. Reduced samples of FNR and Fld for stopped flow were prepared by photoreduction with 5-dRf as TABLE I Midpoint reduction potentials of the different Flds (data from Ref. 11) and steady-state kinetics parameters of WT FNR in the NADPHdependent cytochrome c reductase activity using WT and mutant Flds as electron carrier
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