Resonance Raman spectroscopic study of ferric and ferrous carbon monoxide forms of polyethylene glycolated horseradish peroxidase (HRPPEG) in benzene solution is reported. Ferric HRPPEG in benzene solution assumes a 6-coordinate high spin (6-c HS) heme system, binding water, rather than the usual 5-c structure. Low-frequency vibrations associated with porphyrin deformations and propionate bending (nu(8) and delta(CbetaCcCd)) are upshifted by 5 cm(-1), indicating that a heme propionate group is perturbed. The ferrous carbon monoxide complex of HRPPEG (Fe(II)HRPPEG-CO) exists in benzene as a single 6-c species characterized by a weak Fe(II)-H170 bond and weak distal and proximal H-bonding heme pocket interactions. The structural changes are similar to those observed when an aromatic substrate analogue, benzohydroxamic acid (BHA) binds to HRP in aqueous solution. The structural changes are attributed to the formation of an enzyme-substrate complex between HRPPEG and benzene, a known substrate for HRP. Taken together the resonance Raman spectral data for Fe(III)HRPPEG and Fe(II)HRPPEG-CO in benzene suggest that the heme active site in HRPPEG is significantly affected by substrate binding. Heme active site structural alterations may be attributed to dehydration which disrupts critical H-bonding interactions in both the distal and proximal heme pockets, allows substrate access to the heme iron, and promotes salt bridge formation between the propionate and the distal R38 side chain. These structural changes can explain the surprising observation that HRPPEG can hydroxylate benzene, with the incorporation of peroxide oxygen.