We have measured the binding affinity (K A ) and electron transfer (ET) rate constants (k) for the complex of hemoglobin (Hb) and cytochrome b 5 (b 5 ), using triplet quenching titrations of mixed-metal [ZnM, Fe 3+ (N 3 -)] Hb hybrids and of fully substituted Zn-mesoporphyrin (ZnM)Hb by b 5 (trypsin-solubilized, bovine) (pH values 6.0 and 7.0). The use of the mixed-metal Hb hybrids with Zn in one chain type allows us to selectively monitor the 3 ZnP f Fe 3+ P ET reaction of Fe 3+ b 5 with either the R-chains or the β-chains. The self-consistent analysis of the results for the mixed-metal hybrids and those for the (ZnM)Hb allows us to determine the reactivity and affinity constants for the interactions of b 5 with the individual subunits of T-state Hb. The results confirm that ET occurs within a complex between b 5 and Hb, not through a simple bimolecular collision process. At pH 6.0, the binding affinity constant of the R-chains (K R ≈ 2.0 × 10 4 M -1 ) is ∼4-fold larger than that of the β-chains (K β ) 4.9 × 10 3 M -1 ); the intracomplex ET rate constant of the R-chains (k R ≈ 1500 s -1 ) is ∼2-fold larger than that of the β-chains (k β ≈ 850 s -1 ). The binding affinity and ET rate constant of the R-chains both decrease as the pH is increased from 6.0 to 7.0; the binding affinity of the β-chains is essentially the same at pH 6.0 and 7.0, while the ET reactivity decreases. The kinetic results are consistent with a docking model in which each subunit binds a molecule of b 5 . However, they permit an alternative in which b 5 reacts with the R-chains by binding at a site which spans the R 1 β 2 dimer interface.
Sarcosine oxidase from Corynebacterium sp. P-1 is a heterotetrameric enzyme (alphabetagammadelta) that contains two noncovalently bound coenzymes (FAD, NAD+) and covalently bound FMN [8alpha-(N3-histidyl)FMN] which is attached to the beta subunit. Chlumsky et al. [(1995) J. Biol. Chem. 270, 18252-18259] tentatively identified His175 as the covalent FMN attachment site in the beta subunit, based on an alignment of the sequence of C. sp. P-1 beta subunit with a highly homologous flavin-containing peptide from another corynebacterial sarcosine oxidase (C. sp. U-96). To test this hypothesis, His175 in the C. sp. P-1 beta subunit was mutated to an alanine. Unexpectedly, the mutant enzyme was found to contain 1 mol of covalently bound flavin and to exhibit catalytic activity similar to wild-type enzyme. Covalent flavin-containing peptides were isolated from wild-type and mutant enzymes and analyzed by electrospray mass spectrometry. The mass observed for the mutant peptide (1152.4 Da) matched that predicted for an FMN-containing hexapeptide, corresponding to residues 173-178 (1152.1 Da). In the mutant, this region (HDAVAW) contains a single histidine (His173) which must be the covalent flavin attachment site. The mass observed for the wild-type peptide (1218.6 Da) matched that predicted for an FMN-containing hexapeptide, also corresponding to residues 173-178 in the beta subunit (1218.2 Da). This region in the wild-type enzyme includes two histidine residues (HDHVAW). Attempts to sequence the wild-type or mutant peptides by automated Edman degradation were unsuccessful. Instead, the peptide sequences were investigated by collisional-activated dissociation (CAD) and tandem mass spectrometry. The CAD mass spectral data with the mutant peptide confirmed the sequence deduced based on the mass of the intact peptide. The CAD mass spectral results with the wild-type peptide showed that FMN was covalently attached to the N-terminal histidine in the hexapeptide, which corresponds to His173 in the beta subunit.
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