Abstract
Truncated hemoglobins (trHbs), are a distinct and newly characterized class of small myoglobin-like proteins that are widely distributed in bacteria, unicellular eukaryotes, and higher plants. Notable and distinctive features associated with trHbs include a hydrogen-bonding network within the distal heme pocket and a long apolar tunnel linking the external solvent to the distal heme pocket. The present work compares the geminate and solvent phase rebinding kinetics from two trHbs, one from the ciliated protozoan Paramecium caudatum (P-trHb) and the other from the green alga Chlamydomonas eugametos (C-trHb). Unusual kinetic patterns are observed including indications of ultrafast (picosecond) geminate rebinding of CO to C-trHb, very fast solvent phase rebinding of CO for both trHbs, time-dependent biphasic CO rebinding kinetics for P-trHb at low CO partial pressures, and for P-trHb, an increase in the geminate yield from a few percent to nearly 100% under high viscosity conditions. Species-specific differences in both the 8-ns photodissociation quantum yield and the rebinding kinetics, point to a pivotal functional role for the E11 residue. The response of the rebinding kinetics to temperature, ligand concentration, and viscosity (glycerol, trehalose) and the viscosity-dependent changes in the resonance Raman spectrum of the liganded photoproduct, together implicate both the apolar tunnel and the static and dynamic properties of the hydrogen-bonding network within the distal heme pocket in generating the unusual kinetic patterns observed for these trHbs.
Highlights
MethodsDescribed methods were used to generate and prepare samples of P-trHb (5) and C-trHb (2)
The kinetic patterns observed for the recombination of CO to trHbs are varied and atypical when compared with many other Hbs and Mbs
The observed patterns for the two trHbs studied in the present work can largely be explained based on residuespecific variations in the B10Y-based hydrogen-bonding network within the distal heme pocket and the presence of a long apolar tunnel linking the distal heme pocket and the solvent
Summary
Described methods were used to generate and prepare samples of P-trHb (5) and C-trHb (2). Samples of Mb(L29W) and Mb(H64Q/L29Y) were a generous gift from Professor John Olson of Rice University. Geminate and solvent phase recombination measurements were carried out using 8 ns 532-nm pulses at 1 Hz from a Nd:YAG laser (Minilite, Continuum, Santa Clara, CA) as a photodissociation source and a greatly attenuated continuous wave 442-nm probe beam to monitor time-dependent changes in absorption. Data collection, and data display can be found in a previous publication and citations therein (41). The Soret enhanced resonance Raman spectra of the photoproducts of several of the samples was generated using 8 ns 435.8-nm excitation pulses to both photodissociate the CO derivatives and generate the Raman spectrum of the resulting photoproduct. Data collection, and data analysis are reported elsewhere (44, 45)
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