pH and Driving Force Dependence of Intramolecular Oxyferryl Heme Reduction in Myoglobin

Abstract

The kinetics of oxyferryl (Fe IV dO) heme reduction in horse heart myoglobin (Mb) by a 4 LRu II (a ) NH 3 ; L ) NH 3 , pyridine, isonicotinamide) bound at the surface His48 were investigated with pulse radiolysis. The observed first-order rate constants (k obs1 ) decreased with increasing pH and reduction potential for the a 4 LRu centers (E°/ Ru III/II) 77, 330, and 400 mV for L ) NH 3 , Pyr, and Isn). Rate-pD data obtained in D 2 O for the a 5 Ru derivative revealed the presence of an equilibrium isotope effect, and a pK a of 5.7 (6.2 in D 2 O) was obtained for the acid-base group, which is assigned to the distal His64. A mechanism where protonation precedes ET provides a good fit of the kinetic data for the three a 4 LRu derivatives. Marcus theory analysis of the k ET (0.74, 1.8, and 3.6 s -1 for L ) Isn, Pyr, and NH 3 ) extracted from the k obs1 values yielded a reorganization energy (λ) of 1.8 for Ru II f Fe IV dO ET in the a 4 LRu derivatives but a λ of 2.1 eV for the a 5 Ru derivative. From the latter, it is concluded that ET is strongly gated in the a 5 Ru derivative, and this is assumed to be the major reason for the low reactivity of Fe IV dO in Mb at high -∆G°.

Crystallographic and Spectroscopic Studies of Peroxide-derived Myoglobin Compound II and Occurrence of Protonated FeIV–O

“…Several studies have proposed a pH dependence of compound II in Mb (39 -41, 111), and a low and high pH form with different absorption spectra is observed at pH 6 and 8.6, respectively (41). However, for Mb the 797 cm Ϫ1 oxoferryl rRaman mode is insensitive to pH changes in the range from 6 to 12 (112,113). From our crystallographic Mb compound II studies we were not able to detect any change in the Fe-O distance in the pH range 5.2-8.7 nor any differences in single-crystal light absorption spectra (Figs.…”

Catalytic effect of ferricyanide between myoglobin and luminol and effect of temperature

“…Although there are a few instances concerning immunoassay of Mb, reports on the application of Mb in CL analysis are few (8). Recently, we reported that Mb and luminol could react directly, which was also shows, the mixture of equimolar K 3 Fe(CN) 6 and Mb reacted with luminol and produced very strong CL emission, far more intense than that of Mb-luminol or the K 4 Fe(CN) 6 -luminol system.…”

“…It was reported that in the haem pocket of Mb(Fe III ), iron is in the ferric state and has a considerable positive redox potential, E° (Mb(Fe III )/ MbO 2 ) = 0.34 V (11), while ferricyanide E° (Fe(CN) 6 3− / Fe(CN) 6 4− ) = 0.36 V. Thus, in the presence of ferricyanide in solution, Fe(CN) 6 3− could oxidize MbO 2 , producing metmyoglobin Mb(Fe III ), in which the iron is ferric, and Fe(CN) 6 4− . It was reported that (Fe(CN) 6 ) 4− ions could bind to Mb specifically and catalyse electron transfer of the protein (12), and it binds on Mb in the His 119 (GH1) region (13). This was confirmed by the fact that the competitive binding of redox-inactive zinc ion on Mb significantly inhibits the effect of ferrocyanide.…”

“…This was confirmed by the fact that the competitive binding of redox-inactive zinc ion on Mb significantly inhibits the effect of ferrocyanide. Thus, the electron transfer between Mb(Fe III ) and luminol may be accelerated through the formation of the [Mb-Fe(CN) 6 4− -luminol] ternary complex. The possible CL mechanism might involve a quick and efficient electron transfer process after the specific binding of (Fe(CN) 6 ) 4− to Mb in the distal His 119 (GH1) region, catalysing the CL reaction.…”

“…Thus, the electron transfer between Mb(Fe III ) and luminol may be accelerated through the formation of the [Mb-Fe(CN) 6 4− -luminol] ternary complex. The possible CL mechanism might involve a quick and efficient electron transfer process after the specific binding of (Fe(CN) 6 ) 4− to Mb in the distal His 119 (GH1) region, catalysing the CL reaction. Therefore, the whole process could be outlined as following equilibrium:…”