Laser flash photolysis studies of electron transfer to the cytochrome b5-cytochrome c complex

Abstract

Rate constants for electron transfer in the complex between recombinant rat mitochondrial outer membrane cytochrome b5 or the tryptic fragment of bovine liver cytochrome b5 and horse mitochondrial cytochrome c were measured by laser flash photolysis of 5-deazariboflavin-EDTA solutions. When an excess of cytochrome b5 was titrated with increasing amounts of cytochrome c at low ionic strength and electron transfer was initiated by a laser flash, both proteins were rapidly reduced by deazariboflavin semiquinone. The initial photoreduction was followed by a slower second-order reduction of b5 complexed oxidized cytochrome c by free reduced cytochrome b5. At an 8:1 ratio of cytochromes b5 to c, the pseudo-first-order rate constant for reduction of complexed cytochrome c increased 3-5-fold between ionic strengths of 5 and 40 mM, and then dropped precipitously at higher ionic strengths. The ionic strength dependent increase in rate constant is likely to be due to relief of steric hindrance via rearrangement of cytochrome c in the complex. The reaction rate showed no sign of saturation at any ionic strength, indicating a first-order rate constant greater than 10(4) s-1 within a transient ternary protein complex; i.e., interprotein electron transfer approaches the largest values previously reported for the stable binary protein complex (approximately 4 x 10(5) s-1). Our results emphasize the flexibility of electron-transfer protein complexes, which had previously been modeled in a single conformation with specific salt bridges. It appears that a variety of orientations can exist within such protein-protein complexes and that the population of conformations changes with ionic strength.(ABSTRACT TRUNCATED AT 250 WORDS)

A “parallel plate” electrostatic model for bimolecular rate constants applied to electron transfer proteins

“…We have generally used a value of 10 for this parameter. Charge products that reflect the local charge at the presumed sites of electron transfer for reactions between cytochrome c and flavodoxin or cytochrome bS have been obtained using this procedure Meyer et al, 1993b). These values are comparable to those calculated for reaction with the small molecule reductant FMN semiquinone, for which the charge is easier to estimate .…”

“…protein-protein electron transfer rate constants (Meyer et al, , 1987(Meyer et al, , 1993a(Meyer et al, , 1993b(Meyer et al, , 1993cTollin et al, 1984Tollin et al, , 1986Tollin et al, , 1993Przysiecki et al, 1985;Cheddar et al, 1986Cheddar et al, , 1989. In all cases where the experimental data show a monotonic increase or decrease in rate constant with ionic strength, the rate constant versus square root of ionic strength curves can be fit using the parallel plate model monopole term (K;) only.…”

“…There are several examples of more complicated ionic strength dependencies for electron transfer protein reactions. These either show an increase in rate constant at low ionic strengths and then a decrease at higher ionic strengths (Hazzard et ai., 1988(Hazzard et ai., , 1991Walker et al, 1991;Zhou & Kostic, 1992;Hurley et al, 1993;Meyer et al, 1993bMeyer et al, , 1993cPan et al, 1993;Tollin et al, 1993), or the opposite dependence, i.e., a decrease followed by an increase in rate constant with increasing ionic strength (Cheddar et al, 1989). Such data are more difficult to analyze than are the simple cases, but can be fit with the parallel plate model using both monopolar and dipolar terms, as shown in Figure 5.…”

Refinement and structural analysis of bovine cytochrome b5 at 1.5 Å Resolution

“…The solvent-exposed heme propionate and negatively charged residues on the cytochrome b5 surface, specifically Glu43, Glu44, Glu48 and Asp60 were the first residues identified as forming linkages in these complexes. Both kinetic and NMR studies of the bs-c complexes (Whitford, Concar, Veitch & Williams, 1990;Meyer et al, 1993) confirmed that ionic strength modulates the formation and stability of the binary interactions and showed that there was considerable rotation of the partners in the complex.…”

Negatively Charged Anabaena Flavodoxin Residues (Asp144 and Glu145) Are Important for Reconstitution of Cytochrome P450 17α-Hydroxylase Activity

“…Alternatively, elimination of either the Asp 144 or Glu 145 carboxyl group may result in a flavodoxin⅐P450c17 complex whose conformation is more rigid and consequently less favorable for electron transfer relative to wild-type Fld. The need for flexibility of electron transfer complexes has been proposed for other redox proteins including cyt c/cyt c peroxidase (30), cyt c/cyt c oxidase (31), cyt c/cytochrome b 5 (32), and Anabaena ferredoxin/FNR (33). As evidenced by the almost 4-fold increase in apparent K s , replacement of Asp 126 with alanine may indirectly enhance charge repulsion, although not significantly enough to affect electron transfer.…”