Kinetics of CO and O2 binding to human serum albumin-heme hybrid

The kinetics of the CO and O-2 binding to the synthetic hemoprotein, recombinant human serum albumin (rHSA) incorporating eight 2-[8-(N-(2-methyIimidazolyl))octanoyloxymethyl]-5,10,15,20-tetrakis(o-pivalamido)phenylporphinatoiron(II)s(FePs) [rHSA-FeP(8)] have been investigated by laser flash photolysis. Time dependence of the absorption change accompanied the CO rebinding to rHSA-FeP(8) was composed of three phases. The fastest component was the axial base elimination, and the long-lived biphasic decay corresponds to the direct recombination of CO to the five-N-coordinated FePs in rHSA. The rate constants of the fast and slow phases of the CO association [k(on)(CO)(fast), k(on)(CO)(slow)] were determined to be 4.9 x 10(6) M-1 s(-1) and 6.7 x 10(5) M-1 s(-1), respectively. The initial amplitude after the laser pulse gave the concentration ratio of the fast and slow phases (n = 3); (i) two of the eight FePs exhibited the slow rate constants and (ii) they are presumably accommodated in the second and fifth binding sites of FeP in the albumin structure. The absorption decay following the O-2 photodissociation of rHSA-FeP(8) also showed the same behavior. Thermodynamically, the large DeltaG(double dagger) of the slow phase of the CO rebinding, which mainly comes from the enthalpic factor, suggests the appearance of additional steric hindrance on the central metal iron of FeP. Furthermore, orientation of the porphyrin plane in rHSA was predicted by molecular simulation, which supports the experimental data from the kinetic observations.