Dynamics of Ultrafast Rebinding of CO to Carboxymethyl Cytochrome c

Abstract

Rebinding dynamics of CO to carboxymethyl cytochrome c (Ccytc), a chemically modified cytochrome c to bind ligands in its ferrous form, in D(2)O solution at 283 K after photodeligation, was investigated using femtosecond vibrational spectroscopy. The stretching mode of CO bound to the protein shows four stretching bands near 1962 cm(-1). Time-resolved spectra of the bound CO revealed a slight band-position-dependent rebinding kinetics, suggesting that the geminate rebinding of CO depends on the conformation of the protein. The overall rebinding kinetics of CO to Ccytc was more than 1000 times faster than that to myoglobin (Mb), a ligand-binding protein, and is also faster than a model heme, microperoxidase-8 in viscous solvent. The efficient rebinding of CO to Ccytc was attributed to the longer retention of the dissociated CO near the active binding site by the organized protein matrix of Ccytc. The spectra of the dissociated CO reveal a fast-growing band in the picosecond time scale that is assigned to CO in D(2)O solvent. The ultrafast CO escape to bulk solution is consistent with its 3D structure showing a sizable opening in the active site. It appears that most of the dissociated CO rebinds within 1 ns, except for those that escape to the bulk solution through the opening. The CO rebinding in Ccytc indicates that the primary heme pocket in Mb, located near the active site and holding the dissociated ligand for longer than tens of nanoseconds, has a specific structure to suppress CO rebinding.

Dynamics of the Heme-binding Bacterial Gas-sensing Dissimilative Nitrate Respiration Regulator (DNR) and Activation Barriers for Ligand Binding and Escape

“…As CO binds to heme in a nearperpendicular configuration, the CO orientation with respect to the heme normal is expected to be a critical parameter. The barrierless binding rate constants deduced within several protein systems presumably reflect distinct heme-CO bound configurations that interconvert slowly on the time scale of the experiment as have been observed in time-resolved infrared experiments in CooA (46) and carboxymethylated cytochrome c (47).…”

“…Cyt c is a relatively rigid protein (35,36) and does not contain a native ligand binding pocket or ligand access pathway; consistently, the CO escape yield of CO-binding cytochrome c variants is relatively low (17,23,47). Consequently, protein motions are expected to enhance the probability of CO escape.…”

Picosecond Dynamics of CN--Ligated Ferric Cytochrome c after Photoexcitation Using Time-resolved Vibrational Spectroscopy

“…3,9,31 Two optical parametric amplifiers (OPA) were used to generate a pump pulse at 575 nm with 3 μJ of energy and a tunable mid-IR probe pulse. 32 The polarization of the probe pulse was set parallel or perpendicular to that the pump pulse to obtain the desired polarized absorbance spectra.…”

“…[1][2][3][4] The binding of a small ligand to heme proteins has been greatly studied in order to understand the relationships between protein structure, dynamics, and function. 2,3,[5][6][7][8][9][10][11][12][13][14] In particular, the rebinding dynamics of a number of neutral diatomic molecules, such as CO, NO, or O 2 to ferrous heme or heme proteins has been widely investigated both experimentally and theoretically. By contrast, only a few experiments have been carried out on the rebinding dynamics of ionic molecules to heme proteins.…”

Unidirectional Photo-induced Charge Separation and Thermal Charge Recombination of Cofacially Aligned Donor-Acceptor System Probed by Ultrafast Visible-Pump/Mid-IR-Probe Spectroscopy

“…[25][26][27] Briefly, two identical home-built optical parametric amplifiers (OPAs), pumped by a commercial Ti:sapphire regenerative amplifier (Hurricane, Spectra Physics) with a repetition rate of 1 kHz, are used to generate a visible pump pulse and a mid-IR probe pulse. Pump pulse at 575 nm with 3.0 mJ of energy was generated by frequency doubling of a signal pulse of one OPA.…”