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BIOPHYSICS
Ligand migration pathway and protein dynamics in myoglobin: A time-resolved crystallographic study on L29W MbCO

Marius Schmidt ^*, , Karin Nienhaus , Reinhard Pahl , Angela Krasselt ^*, Spencer Anderson , Fritz Parak ^*, G. Ulrich Nienhaus ^, ¶, and Vukica rajer 

^*Physikdepartment E17, Technische Universität München, James Franck Strasse, 85747 Garching, Germany; Department of Biophysics, University of Ulm, Albert-Einstein-Allee 11, 89081 Ulm, Germany; Consortium for Advanced Radiation Sources, University of Chicago, 920 East 58th Street, Chicago, IL 60637; and ^¶Department of Physics, University of Illinois at Urbana–Champaign, 1110 West Green Street, Urbana, IL 61801

Communicated by Hans Frauenfelder, Los Alamos National Laboratory, Los Alamos, NM, June 14, 2005 (received for review February 22, 2005)

By using time-resolved x-ray crystallography at room temperature, structural relaxations and ligand migration were examined in myoglobin (Mb) mutant L29W from nanoseconds to seconds after photodissociation of carbon monoxide (CO) from the heme iron by nanosecond laser pulses. The data were analyzed in terms of transient kinetics by fitting trial functions to integrated difference electron density values obtained from select structural moieties, thus allowing a quantitative description of the processes involved. The observed relaxations are linked to other investigations on protein dynamics. At the earliest times, the heme has already completely relaxed into its domed deoxy structure, and there is no photodissociated CO visible at the primary docking site. Initial relaxations of larger globin moieties are completed within several hundred nanoseconds. They influence the concomitant migration of photodissociated CO to the Xe1 site, where it appears at 300 ns and leaves again at 1.5 ms. The extremely long residence time in Xe1 as compared with wild-type MbCO implies that, in the latter protein, the CO exits the protein from Xe1 predominantly via the distal pocket. A well-defined deligated state is populated between 2 µs and 1 ms; its structure is very similar to the equilibrium deoxy structure. Between 1.5 and 20 ms, no CO is visible in the protein interior; it is either distributed among many sites within the protein or has escaped to the solvent. Finally, recombination at the heme iron occurs after >20 ms.

kinetics | Laue crystallography | protein relaxation

Abbreviation: Mb, myoglobin.

Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org [PDB ID codes 2bw9 (Mb^RT) and 2bwh (MbCO^L)].

To whom correspondence should be addressed. E-mail: marius.schmidt{at}ph.tum.de.

© 2005 by The National Academy of Sciences of the USA

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