Ligand Dynamics in an Electron Transfer Protein

Abstract

Substitution of the heme coordination residue Met-80 of the electron transport protein yeast iso-1-cytochrome c allows external ligands like CO to bind and thus increase the effective redox potential. This mutation, in principle, turns the protein into a quasi-native photoactivable electron donor. We have studied the kinetic and spectral characteristics of geminate recombination of heme and CO in a series of single M80X (X ‫؍‬ Ala, Ser, Asp, Arg) mutants, using femtosecond transient absorption spectroscopy. In these proteins, all geminate recombination occurs on the picosecond and early nanosecond time scale, in a multiphasic manner, in which heme relaxation takes place on the same time scale. The extent of geminate recombination varies from >99% (Ala, Ser) to ϳ70% (Arg), the latter value being in principle low enough for electron injection studies. The rates and extent of the CO geminate recombination phases are much higher than in functional ligand-binding proteins like myoglobin, presumably reflecting the rigid and hydrophobic properties of the heme environment, which are optimized for electron transfer. Thus, the dynamics of CO recombination in cytochrome c are a tool for studying the heme pocket, in a similar way as NO in myoglobin. We discuss the differences in the CO kinetics between the mutants in terms of the properties of the heme environment and strategies to enhance the CO escape yield. Experiments on double mutants in which Phe-82 is replaced by Asp or Gly as well as the M80D substitution indicate that such steric changes substantially increase the motional freedom-dissociated CO.

Ultrafast photochemistry of the bc1complex

“…These studies involved mostly dynamics of external ligands that can bind as competitive inhibitors, and also methods devised to use ligand dissociation for studying, with high time resolution, electron transfer within cytochrome c oxidase 7, 8 and between cytochrome c and cytochrome oxidase. 9, 10 . Until recently, such studies were not available for the isolated cytochrome bc 1 complex, which binds external ligands only to a very limited extent, and not in a functional manner.…”

Ligand Dynamics and Early Signaling Events in the Heme Domain of the Sensor Protein Dos from Escherichia coli

“…5B) of competition between essentially barrierless (Ͻ10 meV) rebinding ( Ϸ 2.8 ns) and activated (barrier enthalpy 45 Ϯ 10 meV) migration of CO out of the direct heme environment with a similar rate around room temperature. The 2.8-ns rebinding phase is substantially slower than the fastest phases of CO rebinding observed in several other heme proteins (19,39,40) that are on the sub-100-ps timescale. Therefore, we suggest that this phase does not reflect the intrinsic heme-CO rebinding rate, but rather is limited by very low barrier motions of CO in the heme pocket in adopting a favorable configuration for rebinding.…”

Nitric Oxide Binds to the Proximal Heme Coordination Site of the Ferrocytochrome c/Cardiolipin Complex

“…The spectral changes accompanying NO binding indicate that the primary binding process leads to a species that is similar to the final NO bound form and different from the NO adducts of cm cyt. c or the M80X mutants that we have previously described (22) and shown to be hexacoordinate complexes (Fig. 1).…”

“…Kinetic analysis yields a second order rate constant k 1 of 2 ϫ 10 7 M Ϫ1 s Ϫ1 , very much in line with rate constants of NO binding to many other heme proteins, but interestingly orders of magnitude higher than binding to AXCP (24) and ferro M80A cyt. c (22) in which binding is rate limited by steric hindrance provided by the tightly packed protein cage. The spectral changes accompanying NO binding indicate that the primary binding process leads to a species that is similar to the final NO bound form and different from the NO adducts of cm cyt.…”

“…Multicolor femtosecond absorption spectroscopy using 60-fs pulses centered at 560 nm and white light continuum probe pulses in the 380 -480 nm region, at a repetition rate of 30 Hz, and data analysis was performed as described (22). Stopped-flow spectroscopy was carried out using an Applied Photophysics SX-20 instrument.…”